Pathogen detection biosensor

ABSTRACT

The invention described herein provides methods for the detection of target particles, such as pathogens, soluble antigens, nucleic acids, toxins, chemicals, plant pathogens, blood borne pathogens, bacteria, viruses and the like. Also described is an emittor cell comprising a receptor, wherein the receptor can be an antibody or an Fc receptor, and an emittor molecule for the detection of a target particle in a sample wherein the target particle to be detected is bound by one or more receptors on the emittor cell. Also provided are optoelectronic sensor devices for detecting a target particle in a sample, including in a plurality of samples.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/085,495, now allowed, which has a 371(c) date of Mar. 27, 2009, nowU.S. Pat. No. 8,216,797 which is the U.S. National Stage ofInternational Application No. PCT/US2006/045691, filed Nov. 30, 2006,published in English, which is a continuation-in-part of U.S.application Ser. No. 11/001,583, filed Dec. 1, 2004, now U.S. Pat. No.7,422,860, issued Sep. 9, 2008, which is a continuation-in-part of U.S.application Ser. No. 10/467,242, filed Jan. 16, 2004, now U.S. Pat. No.7,214,346, issued May 8, 2007, which is the U.S. National Stage ofInternational Application No. PCT/US02/03606, filed Feb. 6, 2002,published in English, which claims the benefit of U.S. ProvisionalApplication No. 60/266,977, filed Feb. 7, 2001.

The International Application No. PCT/US2006/045691, filed Nov. 30,2006, also claims the benefit of U.S. Provisional Application No.60/741,271, filed on Nov. 30, 2005.

The entire teachings of the above applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

This invention was made with Government funds from U.S. Air Forcecontract no. F19628-00-C-0002. The Government has certain rights in theinvention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

a) File name: 00502091015SEQ.txt; created Apr. 24, 2012, 5.28 KB insize.

BACKGROUND OF THE INVENTION

The need for small, fast, and sensitive detectors of biological agentswhich are able to monitor an environment for extended periods of time isunderscored by the proliferation of biological and chemical weapons, thepoor man's nuclear weapon. Under battlefield conditions, a usefuldetector would rapidly alert a soldier when a specific biological orchemical agent is detected so that countermeasures can quickly beimplemented.

Such detectors would be useful in non-military applications as well.Rapid detection of antibiotic-resistant bacteria in a patient would helpclinicians select a more effective therapeutic regimen. Continuousmonitoring of a city's drinking water supply would provide early warningof potential pathogens, giving public works officials more time tomanage the potential health risks to the public. In addition, the use ofthese detectors in meat and poultry inspections would be a significantimprovement over the current “poke-and-smell” procedure. In general,such detectors are sorely needed analytical and diagnostic applicationswithin the fields of medicine (e.g., veterinary medicine), agriculture,environmental protection (e.g., to diagnose sick building syndrome), andfood processing or regulation.

All vertebrates acquire a specific immune response to a foreign agent(antigen) in part by generating an immense diversity of antibodymolecules. Antibody molecules bind to antigen with high specificity,e.g., they can differentially bind to two closely related strains ofbacteria, viruses, protein, nucleic acid, fungus, protozoa,multicellular parasite, or prion, as well as products produced orinduced by those particles.

Antibodies are produced by B cells, a crucial component of the immunesystem. An antigen can activate a B cell by binding to antibodies on itssurface, leading to a cascade of intracellular biochemical reactionswhich causes a calcium ion influx into the cytosol of the B cell.

For a review of antibody structure and function and B cell activation,see Paul, editor, Fundamental Immunology, 3rd ed., Raven Press, New York(1993).

Devices that exploit antibody diversity for detection of multiple andrare target particles or antigens have been described in U.S. Pat. No.6,087,114 and U.S. Pat. No. 6,248,542.

These devices generally include a liquid medium containing sensor cells(e.g., a B cell, macrophage or fibroblast), also referred to herein as“CANARY” cells or “emitter” cells, an optical detector, and the liquidmedium receiving target particles to be detected. Each of the cells hasreceptors (e.g., chimeric or single chain antibodies) which areexpressed on its surface and are specific for the antigen to bedetected. Binding of the antigen to the receptor results in a signalingpathway involving chemical or biochemical changes (e.g., an increase incalcium concentration). The cells also contain emitter molecules (e.g.,aequorin or indo-1) in their cytosol which can emit photons in responseto the signaling pathway (e.g., increased calcium concentration in thecytosol). The detector can be separated from the medium containing thecells by a covering (e.g., glass) that is transparent to the photons.Such a covering can serve to support the medium, protect a fragilesurface of the detector, or be used as a lens. The optical detector,e.g., a charge-coupled device (CCD) is able to detect the photonsemitted from the cells in response to the receptor-mediated signalingpathway and indicate to the user that the antigen to be detected ispresent. Other optical detectors which can be used in the device includephotomultiplier tubes, photodiodes, complimentary metal oxidesemiconductor (CMOS) imagers, avalanche photodiodes, andimage-intensified charge-coupled devices (ICCD) (see for example, thoseavailable from Photek Ltd., East Sussex, UK). In some embodiments, theoptical detector is able to distinguish individual cells.

SUMMARY OF THE INVENTION

Provided herein are methods for the detection of target particles. Inparticular, methods are provided for the detection of biological agents,pathogens, bacteria, viruses, soluble antigens, toxins, chemicals,explosives, nucleic acid sequences (for example, DNA or RNA), plantpathogens, blood borne pathogens, and the like.

Methods of detecting target particles include detection of targetparticles in liquid samples, aerosol samples, and dry samples.

Also provided herein is an emittor cell comprising a receptor, whereinthe receptor can be an antibody specific for a target antigen, anantibody specific for a general target (for example a label such asbiotin, or an immunoglobulin, and the like). In addition, the receptorcan be an Fc receptor.

The emittor cell further comprises an emittor molecule for the detectionof a target particle in a sample wherein binding of the receptor to thetarget particle stimulates a response from the emittor molecule. In oneembodiment, the receptor stimulates an increase in intracellular calciumconcentration, wherein the emittor molecule emits a photon in responseto the increase in intracellular calcium. In one embodiment, the emittormolecule is aequorin. In another embodiment, the emittor molecule is anaequorin-GFP molecule.

Also provided is an optoelectronic sensor device for detecting a targetparticle in a plurality of samples using a photon detector. Anoptoelectronic sensor device can detect a target particle in a liquidsample. Alternatively, an optoelectronic sensor device can detect atarget particle in an air or aerosol sample. In one embodiment, thesensor device comprises centrifugation means. In another embodiment, thesensor device does not comprise a centrifugation means. In oneembodiment, the sensor device comprises and aerosol spray. In anotherembodiment, the sensor device comprises a wicking means. In a furtherembodiment, the sensor device comprises a moveable substrate. In oneembodiment, the sensor device comprises a pinhead substrate for captureof target particles.

Detection of a target particle (such as a soluble antigen or a nucleicacid) is mediated in part by binding of the target particle to areceptor, either directly or indirectly, expressed on the cell surfaceof an emittor cell. Direct binding can be via a receptor, such as anantibody, which binds directly and specifically to the target particle.Indirect binding of the target particle can be through an Fc receptorthat binds to an antibody that has been attached (bound) to the targetparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic of the optoelectronic sensor cellular concept.

FIG. 2 is a schematic showing the general architecture of anoptoelectronic sensor having a sampler (trigger) for preliminary sensingof suspect agents.

FIG. 3 is a schematic illustrating the creation of cell lines for use inthe optoelectronic sensor.

FIG. 4 is a schematic of an integrated biological aerosol warning sensor(BAWS)/optoelectronic sensor system.

FIG. 5 illustrates the B cell response to foot-and-mouth disease virusin the optoelectronic sensor.

FIG. 6 illustrates a dry-impactor module for the optoelectronic sensor.

FIG. 7 is a schematic illustrating the effect of localization andmixing.

FIG. 8 illustrates the effect of localization using tularemia cells.

FIG. 9 illustrates an automated cell-delivery module for theoptoelectronic sensor.

FIG. 10 illustrates a dose response relationship for a sample oftularemia cells using the optoelectronic sensor.

FIG. 11 illustrate B cell resistance to chemical and biologicalcontamination.

FIG. 12 illustrates an automated centrifuge module for theoptoelectronic sensor.

FIG. 13 is a schematic illustrating an air impactor/optoelectronicsensor.

FIG. 14 is a schematic illustrating an optoelectronic sensor.

FIG. 15 illustrates an optics-photomultiplier (PMT) module for theoptoelectronic sensor.

FIG. 16 is a schematic illustrating an air impactor/optoelectronicsensor.

FIG. 17 is a schematic illustrating a multi-channel centrifuge in theoptoelectronic sensor.

FIG. 18 is a schematic illustrating a wet centrifuge/impactor concept inthe optoelectronic sensor.

FIG. 19 is a schematic illustrating a wet centrifuge/impactor concept inthe optoelectronic sensor.

FIG. 20 is a schematic of a custom tube for the optoelectronic sensor.

FIG. 21 illustrates an integrated dry-impactor/optoelectronic sensor.

FIG. 22 illustrates the effect of cell treatments on the response ofYersenia pestis specific B cells.

FIG. 23 illustrates an impactor configured to collect aerosol samples.

FIG. 24 is a schematic overview of the concept underlying the “CANARY”sensor. B cells have been modified such that they express aequorinwithin the cell interior and antibodies to pathogen on the cell surface.In the presence of pathogen, the antibodies are “crosslinked”(immobilized, aggregated) on the surface of the cell, stimulating asignaling cascade that results in increased intracellular calcium.Aequorin responds to this increase in intracellular calcium by oxidizingaequorin, and emitting light. Photon output can be monitored using aPMT.

FIG. 25 is a schematic of DNA detection. Oligonucleotides complementaryto target DNA sequences and containing a terminal digoxigenin label arehybridized to the target DNA. Multiple digoxigenin-labeledoligonucleotides bound along the target DNA bind to digoxigeninantibodies on the surface of the CANARY cell (emittor cell), stimulatinglight emission.

FIGS. 26A-C are graphs. CANARY cells (emittor cells) expressingantibodies against digoxigenin can be stimulated by digoxigenin-labeledDNA. Emittor cells expressing antibody against digoxigenin were added tocentrifuge tubes containing 50 μl of the indicated concentration ofdigoxigenin-labeled DNA standards. The tube was centrifuged briefly topellet the cells at the bottom of the tube, nearest the PMT, and photonemission as a function of time recorded. Three different types ofdigoxigenin-labeled DNA were used to stimulate the cells, and each wassuccessful with a different degree of sensitivity. FIG. 26A. Plasmid DNAdensely labeled with digoxigenin (approximately 4000 base pairs with 200digoxigenin molecules attached) was detected with a limit of detectionof approximately 1 ng/ml (50 pg absolute). FIG. 26B. DNA molecularweight standards of various sizes (81-8576 base pairs) sparsely labeledwith digoxigenin (once per 200 base pairs) were detected at 1 μg/ml (50ng absolute). FIG. 26C. DNA molecular weight standards of various sizes(8-587 base pairs) each labeled with 2 digoxigenins (one digoxigenin oneach end of the DNA molecule) were detected at 100 ng/ml (5 ngabsolute).

FIGS. 27A-B are graphs. Centrifugation of cells may decrease sensitivityto soluble antigen. Emittor cells expressing antibody againstdigoxigenin were added to centrifuge tubes containing 50 μl of theindicated concentration of digoxigenin-labeled plasmid DNA. FIG. 27A.The tubes were centrifuged briefly to pellet the cells at the bottom ofthe tube, nearest the PMT, and photon emission as a function of timerecorded. FIG. 27B. The cells and DNA were mixed manually and placedover the PMT without centrifugation.

FIG. 28 is a graph. Two complementary Dig-labeled oligonucleotides(Oligo “3” and Oligo “NEG 3”) were allowed to hybridize underexperimental conditions. The sample was diluted 1:10 with CO2I media toa total volume of 100 μl, 20 μl of cells were added, and light emissionmeasured. Dig cells express the Dig antibody, while control cells donot.

FIG. 29 is a graph. Rapid hybridization of Digoxigenin labeled, ssDNAoligonucleotides. The indicated amount of oligonucleotide “NEG3” wasadded to 8 μl of hybridization solution (50 mM NaCl, 40 mM Tris pH 7.5).1 μl of “oligo 3” was added, followed immediately by 90 μl of CO2Imedium and 20:1 of Dig CANARY cells. The tube flicked to mix, quicklyplaced into the luminometer and light output monitored. The total timebetween addition of the second oligonucleotide and placement in theluminometer (“0” on the x axis) was approximately 15 seconds.

FIG. 30 is a graph. Single stranded DNA was generated from thepBluescript phagemid, and hybridized to all 10 Dig-labeledoligonucleotides. After hybridization the reaction was diluted to 100 μlin CO2I, 20 μl of Dig cells were added, and light emission was measured.The molar ratio indicated in the legend is that of oligonucleotide totarget ssDNA. The ideal ratio in this experiments appears to be between1:2 and 1:4.

FIG. 31 is a graph. Sequence-specific detection of single-stranded DNA.Ten digoxigenin-labeled oligonucleotide probes complementary to the (+)strand of phagemid pBluescript were hybridized to the indicated amountof single-stranded phagemid DNA. Emittor cells expressing antibody todigoxigenin were added, and light output from the cells monitored on aphotomultiplier tube. Only the (+) strand of the phagemid was detected,indicating that the identification is sequence specific. In the absenceof oligonucleotide probe, single-stranded DNA did not stimulate thecells. The limit of detection in this experiment was 50 ng.

FIGS. 32 A-B are bar charts. Effects of hybridization temperature onnucleic acid detection. Single-stranded phagemid DNA was hybridized tothe indicated concentrations of probe at several temperatures, andmaximum RLU plotted. FIG. 32A. Hybridization in PBS shows maximum signalwith hybridization at 51EC, but similar signals from samples hybridizedat 47° C. and 42° C. FIG. 32B. Hybridization at 42° C. displays anincrease in the signal from experiments using lower concentrations ofoligonucleotide probe, such that 0.16 pmoles of oligonucleotide worksnearly as well as 0.63 pmoles, and the signal from 0.04 pmoles wasdoubled.

FIG. 33 is a schematic of the strategy for sedimentation of DNA. Captureoligonucleotides are attached to the surface of a sedimentable particle.These oligonucleotides bind to a region separate from that to which theDig-oligonucleotides bind. Target NA bind to the captureoligonucleotides, and digoxigenin labeled oligonucleotides bind to thetarget. The entire complex is sedimented by centrifugation (or magneticfield), and detected by emittor cells expressing antibody againstDigoxigenin.

FIG. 34 is a graph. Sedimentation of target DNA improves sensitivity.Streptavidin-conjugated beads were saturated with biotin labeled captureoligonucleotide, and excess oligonucleotide removed by washing.pBluescript ssDNA (+strand) was incubated with the beads for 5 min at47° C. and washed. Dig labeled detection oligonucleotides were added,hybridized for 20 min at 47° C., and excess removed by washing. Beadswere resuspended in 200 ul CO2I, and 40 ul used in each assay.

FIG. 35 is a bar chart. pBS phagemid ssDNA was incubated withbiotin-labeled oligonucleotide bound to streptavidin-coated polystyrenebeads and digoxigenin-labeled oligonucleotides for 20 minutes at 47° C.in the indicated concentrations of blocking reagent. The bead bound,digoxigenin labeled target was washed 3 times in TBS (50 mM Tris, 130 mMNaCl) at room temperature. Beads were resuspended in CO2I medium,emittor cells added, and the reaction spun and light output monitored ina luminometer.

FIGS. 36A-C are graphs. FIG. 36A U937 cells exhibit an increase in FcγRI expression when treated with IFN(. The relative expression of Fc (RIon U937 cells treated with IFN ((200 ng/ml, open green peak) oruntreated (solid purple peak) was measured by immunofluorescence. FIG.36B U937 cells express functional aequorin protein. U937 cellstransfected with the calcium-sensitive luminescent protein aequorin emitlight when treated with ionomycin (50 M). FIG. 36C Light is detectedfollowing the crosslinking of the Fc receptors on U937 cells with stableaequorin expression. U937 cells were preincubated with 10 μg/ml humanIgG, then washed and treated with goat anti-human IgG (Fab2′).

FIGS. 37A-D are graphs. U937 cells can be engineered rapidly to respondto several different pathogens or simulants. U937 cells were treated for24 h with IFN ((200 ng/ml) to increase expression of endogenous Fc (RI,and prepared for the CANARY assay. The cells were then incubated withthe following antibodies: FIG. 37A mouse anti-B. anthracis spore, FIG.37B rabbit polyclonal anti-B. anthracis spore, FIG. 37C mouse anti-F.tularensis, or FIG. 37D mouse anti-B. subtilis hybridoma supernatant.Cells were then used in the standard CANARY assay where they detected asfew as 1000 cfu B. anthracis spores with the monoclonal antibody and10,000 cfu spores with the rabbit polyclonal, as well as 10,000 cfu F.tularensis and 1,000 cfu B. subtilis spores.

FIGS. 38A-C are graphs. The rapidly engineered U937 cells are specific,and the specificity is determined by the antibody. FIG. 38A U937 cellsincubated with mouse anti-F. tularensis antibodies did not respond to10⁵ cfu of B. anthracis spores, but did to 106 cfu of F. tularensis.FIG. 38B Cells loaded with mouse anti-B. anthracis spore antibodies didnot respond to F. tularensis but did to 10⁶ cfu of B. anthracis spores.FIG. 38C The cells did not show any response to the 10⁶ cfu of F.tularensis in the absence of anti-F. tularensis antibody [10⁶ cfu F.t.(No ab)].

FIG. 39 is an illustration of a 16-channel sensor. A sensor was designedwhich allowed the simultaneous measurement of 16 samples using a singlelight-gathering channel. The sensor consists of a rotor holding sixteen1.5-ml tubes horizontally, equally distributed about its circumference,and driven by a variable speed motor about a vertical axis. A singlefixed photon-detecting element (e.g., a PMT) is positioned in the planeof the rotor just beyond the path of the tubes during rotation. In thisway, each of the tubes is sequentially and repetitively brought intoclose proximity to the PMT, allowing its light output to be sampled oneach pass. Finally, an optical switch consisting of an optical source(an infrared LED) and a detector (a phototransistor) is used to controlthe counting of detected photons and the reorganization of the data into16 fields, each associated with a specific sample.

FIG. 40 is a graph. Data from the 16-channel sensor demonstrates an LODidentical to that obtained in a single-channel instrument, except that16 samples are measured simultaneously. A single measurement consists ofthe following steps: preparing 16 samples (and/or controls) inindividual 1.5-ml tubes, introducing an aliquot of emittor cells intoeach of the tubes, installing the tubes into the rotor situated in adark box, localizing the emittor cells to the bottom of the tubes usinga brief (5 sec) centrifugal spin at high RCF (˜2000 g), reducing therotor speed to 60 rpm for the duration of the measurement (each tubebeing sampled once every second), and generating a time-series of photoncounts for each sample for display and/or input to a computer algorithmfor evaluation.

FIG. 41 is an illustration of a portable 16-channel-sensor design.

FIG. 42 is an illustration of a CANARY Disc (CD) integrated aerosolcollection and emittor cell delivery.

FIG. 43 is an illustration of an aerosol collection module cutaway withimpaction nozzle and transparent tube.

FIG. 44 is an illustration of an emittor cell delivery module with valvedelivery system.

FIG. 45 is an overview of a 16 channel sensor and results from usingsame.

FIG. 46 is an overview of the detection of toxins.

FIG. 47 is an overview of a sensor cell that expresses aequorin and ageneralized antibody receptor.

FIG. 48 is a schematic for the detection of soluble, monomeric antigens:strategy 1. A single emittor cell is engineered to express two differentantibodies against two different epitopes on the same, monomericantigen. The presence of antigen crosslinks the antibodies, stimulatingthe emittor cell to emit light.

FIG. 49 is a graph depicting the results of a cell line expressingantibodies against both B. anthracis and Y. pestis which was challengedwith each B. anthracis and Y. pestis. This clonal cell line can detectas few as 50 cfu of either B. anthracis and Y. pestis, indicating thatboth antigen-binding sites from both antibodies are expressed andfunctional.

FIG. 50 is schematic of the strategy for detection of soluble proteins.An antigen composed of two or more epitopes is detected using twoantibodies, one bound to beads (or any support that binds to multipleantibodies) and the second antibody is expressed by the emittor cell.The antigen is incubated with the antibody-coated bead, decorating itssurface with multiple antigens. The bead is them presented to theemittor cell. Because the antigen is crosslinked by the bead, theemittor cell antibodies are crosslinked and light emission stimulated.

FIG. 51 is a graph depicting the results of antibody 6E10-10,crosslinked to Protein G magnetic beads, which was incubated withvarying amounts of BoNT/A Hc for 3 hours at 4° C. Beads were washed withCO₂I medium three times. Emittor cells expressing 6B2-2 antibody wereadded, the reaction was spun for 5 seconds, and the light output wasmonitored in a luminometer.

FIG. 52 is a schematic of the detection of a chemical. A peptide isisolated that binds specifically to the chemical of interest, and anantibody generated that binds specifically to the peptide-chemicalcomplex. If the peptide-chemical only forms a single functional epitope,an additional epitope can be incorporated into the peptide. As shown,this epitope is a digoxigenin molecule, but any specific epitope wouldsuffice. In the presence of chemical, the chemical-peptide complex wouldcomprise two antibody-binding sites, and could be detected in a similarmanner as protein toxins.

FIG. 53 is a schematic depicting an alternative method for detecting achemical. Two peptides are isolated that bind in tandem to the chemicalof interest. The binding of these peptides could be detected bygenerating antibodies against each peptide-chemical complex, or bytagging the peptides with antibody binding sites as shown.

FIG. 54 is a schematic of another alternative method for detecting achemical. A peptide that binds to two chemicals of interest is preparedwhich forms a chemical-peptide dimer complex. An antibody is preparedthat binds specifically to the chemical-peptide dimer complex. Thechemical-peptide dimer complex can contain two antibody binding sitessufficient to stimulate emittor cells to increase intracellular calcium,thereby resulting in photon emission by the emittor molecule.

FIG. 55 is a graph. Digoxigenin labeled oligonucleotides were added toRNA in hybridization buffer (40 mM Tris, 130 mM NaCl, 10 mM DTT, RNasinPlus) and incubated for 2 minutes at 47° C. CO2I medium and cells wereadded, the tube was spun for 5 seconds, and light output monitored on aPMT. The limit of detection in this assay was 20 ng. Lack of response tothe control RNA (opposite strand) indicates that the assay is sequencespecific.

FIG. 56 is a schematic. Antigens, including toxins, may be comprised of2 or more epitopes. A CANARY cell typically expresses a single antibodyagainst the target epitope. In a worst case scenario the antigen may bepresent in monomeric form. While the antibody on the CANARY cell iscapable of binding to the monomeric antigen, the antibodies are notcrosslinked and no light is emitted.

FIG. 57 is a schematic. Detection of soluble toxin simulant by CANARY.Monoclonal antibody against one epitope on the BoNT/A Hc protein(6E10-10) were produced from hybridomas, and conjugated to protein-Gcoated magnetic beads. Addition of these beads to solution containingBoNT/A will coat the beads with multiple copies of immobilized antigen,which can stimulate CANARY cells expressing antibody against anonoverlapping epitope.

FIG. 58 is a graph. Protein A-coated beads were conjugated to 6E10-10antibody, and these beads added to BoNT/A Hc diluted in CO2I medium. Thetube was rotated for 2 minutes, CANARY cells expressing 6B2-2 antibodywere added, and the mixture spun for 5 seconds. Light output wasmonitored using a PMT.

FIG. 59 is a bar chart. Effect of frozen storage on BoNT Hc solubilityand antigenicity. 400 ng/ml BoNT Hc was used to challenge the bead-basedCANARY assay. Fresh antigen gives a response 35 fold over background.Frozen and thawed agent gives slightly reduced response, andcentrifuging that frozen-thawed material significantly decreases theresponse, indicating the generation of aggregates during the freeze-thawprocess. A second lot of BoNT Hc from the same company shows lessreactivity, indicating a significant lot-to-lot variation in theantigen.

FIG. 60 is a graph. Detection of BoNT/A Hc in Blood Products. CANARY iscapable of detecting soluble BoNT/A Hc in blood products. BoNT/A Hc (400ng/ml**) spiked into CO2I and incubated with beads for 12 minutes, givesa strong signal. Whole blood spiked with BoNT/A Hc, prior to removal ofcellular material, is also detected by CANARY. BoNT/A Hc spiked intoplasma after removal of blood cells (prepared as described elsewhere)also produces a statistically significant response. **Note that theseassays tested frozen and thawed BoNT/A Hc, so the apparent sensitivitydepicted is adversely affected by the loss of aggregated antigen duringstorage.

FIG. 61 is a graph. Detection of BoNT/A Hc in urine. No samplepreparation was required for detection of BoNT/A in urine samples. Beadswere added directly to the urine spiked with 400 ng/ml** BoNT/A Hc,incubated for 12 minutes, and the beads removed magnetically. Media wasadded to the beads, followed by CANARY cells, and the sample spun for 5seconds. Two of the three spiked urine samples show significant signal,while the signal from the third sample is low. Control urine, which wasnot spiked with BoNT/A Hc prior to bead addition, gives no signal,indicating that nonspecific stimulators are not present. **Note thatthese assays used frozen and thawed BoNT/A Hc, so the apparentsensitivity depicted is adversely affected by the loss of aggregatedantigen lost during storage.

FIG. 62 is a schematic. An alternate sample preparation procedure isused for samples derived from nasal swabs. Sample preparation requiresthe swab itself, a basket containing an integral 5 micron filter, andthe assay tube. The swab is collected, the swab handle trimmed off, andthe swab head is placed into the filter basket. CO2I medium is added tothe swab, and the assembly capped and centrifuged. Beads are added tothe filtrate and the assay carried out without further modification.

FIG. 63 is a graph. Nasal swabs were collected as described, placed intothe filter basket, and CO2I media spiked with BoNT/A Hc at 400 ng/ml**was added. The sample was filtered, beads added, and assayed. Theresponse against these spiked samples was similar to responses againstmock swabs, in which no nasal “material” was present, indicating thatthe nasal secretions prepared in this manner contain no inhibitors.Nasal swabs to which only CO2I was added show no response, indicatingthat nasal swabs do not contain any nonspecific stimulators of theassay. **Note that these assays tested frozen and thawed BoNT/A Hc, sothe apparent sensitivity depicted is adversely affected by the loss ofaggregated antigen during storage.

FIG. 64 is a bar chart. Toxin detection in different liquids. BoNT/A Hcwas spiked into each solution to the indicated concentrations. To 10 μlof that solution was added 1.4 μl of solution containing 560 mM NaCl,1.4 M Hepes pH 7.9, and 6E10-10 conjugated paramagnetic beads. Thesample was rotated for 12 minutes, 190 μl of assay medium added, and thebeads magnetically captured (30 seconds). Unbound material wasdiscarded, and the beads brought up in 50 μl of assay medium. 20 μlCANARY cells expressing antibody against the 6B2-2 epitope were added,the tube spun for 5 seconds, and place in a luminometer. Values are thepeak light output (photons/second) divided by the light output of CANARYcells in medium without antigen (red bar, 0 ng/ml). The wash procedureremoves nonspecific stimulators, as indicated by the similar responsesof the cells to beads incubated in different matrices without antigenaddition. The CANARY assay detects antigen spiked into both orange juice(green bar) and PBT/triton (light blue bar) about as well as controlsolution (assay medium) (red bar), with an LOD of 80 ng/ml. Sensitivityin milk (dark blue bar) is inhibited by greater than 5 fold.

FIG. 65 is a graph. The CANARY assay detects botulinum neurotoxin TypeA. CO2I medium was spiked with the indicated concentrations of botulinumtoxin. 6E10-10 beads were added and incubated for 2 minutes. CANARYcells expressing 6B2-2 antibody were added, the mixture spun for 5seconds, and light output monitored. Samples containing 160 pg of toxin(16 ng/ml) stimulated the cells to more than 10 fold over background.Samples containing 32 pg of toxin (3.2 ng/ml) stimulate cells to morethan 3 fold over background.

FIG. 66 is a graph. CO2I medium was spiked with the indicatedconcentrations of botulinum toxin. Protein G beads conjugated to S25antibody were added and incubated for 2 minutes. CANARY cells expressingRaz antibody were added, the mixture spun for 5 seconds, and lightoutput monitored.

FIG. 67 is a graph. Whole blood was spiked to the indicatedconcentration of BoNT/A. Blood cells were removed by centrifugation, and6E10-10 antibody-coated magnetic beads added to 10 μl of the resultingplasma. The sample was rotated for 2 minutes, 190 ml of medium added,and the tube placed in a magnetic rack for 20 seconds. Media and plasmawas aspirated, the tube removed from the magnet, and 50 μl of CO2I mediaadded. CANARY cells were placed in the cap of the tube and the samplespun for 5 seconds to initiate bead-cell contact, the tube was placed inthe luminometer and light output monitored.

FIG. 68 is a graph. Streptavidin-coated beads were bound to 6E10-10antibody that had been biotinylated with either sulfo-NHS-biotin,sulfo-NHS-LC-biotin or sulfo-NHS-LC-LC-biotin. Beads were added tosolution containing 800 ng/ml BoNT/A Hc, and incubated for 2 minutes.6B2-2 CANARY cells were added, the tube spun for 5 seconds, and lightoutput monitored. Biotin attached by the longest spacer arm (LCLC) gaveslightly better signal.

FIG. 69 is a graph. Biotinylated 6E10-10 antibody bound tostreptavidin-coated beads were added to the indicated concentrations ofBoNT/A and rotated for 15 minutes at room temperature. 6B2-2 CANARYcells were added, the tube spun for 5 seconds, and light outputmonitored on a PMT.

FIG. 70 is a graph. Streptavidin coated magnetic beads were bound to amixture of biotinylated 6C2-4 and 6E10-10 antibody. The beads were addedto the indicated concentrations of BoNT/A and rotated for 15 minutes atroom temperature. 6B2-2 CANARY cells were added, the tube spun for 5seconds, and light output monitored on a PMT.

FIG. 71 is a graph. Beads were diluted 10 fold from the standardconcentration. 100 microliters of 0.32 ng/ml BoNT/A in CO2I at theindicated concentration was added, and the tube rotated overnight atroom temperature. 6B2-2 CANARY cells were added, the tube spun for 5seconds, and light output monitored on a PMT.

FIG. 72 is a schematic.

FIG. 73 is a series of graphs demonstrating U937 cells can be preparedto detect several different pathogens.

FIG. 74 is a pair of graphs demonstrating that U937 aequorin cellsloaded with either monoclonal or polyclonal antibody can detect B.anthracis spore.

FIG. 75 is a summary of the Fc receptor/universal cell line.

FIG. 76 is a summary of an experimental protocol for priming/loadingU937 cells.

FIG. 77 is a summary of the protocol for an Fc receptor cell assay.

FIG. 78 is a summary of alternative embodiments.

FIG. 79 is a summary of alternative embodiments.

FIG. 80 is a series of graphs and a bar chart demonstrating a shift inlight emission for aequorin with EGFP.

FIG. 81 is a series of FACS analysis graphs demonstrating detection ofEGFP fluorescence of aequorin-GFP emittor molecule.

FIG. 82 is a pair of graphs demonstrating detection of aequorin-EGFPwavelength shift by spectrophotometer.

FIG. 83 is a graph demonstrating M12g3R EGFP-aequorin clones functionsimilarly to M12g3R aequorin only cells.

FIG. 84 is a series of graphs demonstrating spore detection by U937aequorin and U937 EGFP-aequorin cells.

FIG. 85 is a series of graphs demonstrating fluorescence of stimulatedCANARY cells.

FIG. 86 is a schematic of multiplex signal detection.

FIG. 87 is a schematic of CANARY cells having different emission colors.

FIG. 88 is a schematic of a cloning strategy for EGFP-aequorin.

FIG. 89 is a schematic of producing pathogen-specific CANARY cells.

FIG. 90 is a schematic of universal CANARY cells.

FIG. 91 is an outline of examples of embodiments of the invention.

FIG. 92 is a schematic for producing universal CANARY cells.

FIG. 93 is a schematic for producing a universal macrophage cell line.

FIG. 94 is a schematic of Fc receptor signaling and production of auniversal sensor cell.

FIG. 95 is a schematic of an anti-Ig (anti-immunoglobulin) universalcell line.

FIG. 96 is a comparison schematic.

FIG. 97 is a summary schematic of universal CANARY cells.

FIG. 98 is an outline of Fc receptor universal cells.

FIG. 99 is an outline of toxin detection embodiments.

FIG. 100 is a graph demonstrating detection of Ralstonia solanacearum ingeranium extract as described herein.

FIG. 101 is a series of photographs illustrating geranium tissueprocessing for pathogen detection.

FIG. 102 is a graph demonstrating detection of polyvirus BYMV using thebead attachment method as described herein.

FIG. 103 is a series of photographs illustrating one embodiment of theapparatus for detection of blood borne pathogens in a blood sample and agraph demonstrating the results of pathogen detection.

FIG. 104 is a schematic illustrating components of the inventiondescribed herein.

FIG. 105 is a graph demonstrating pathogen detection in a blood sample.

FIG. 106 is a graph. Ba Standard with 20 ul cell delivery. 50 ul of Basamples prepared in CO2(I) media and tested with 20 ul B cells. Resultsindicate low background and an LOD of 50 cfu Ba (n=2).

FIG. 107 is a graph. Ba B cell spray. 50 ul of Ba samples prepared inCO2 (I) media and tested with varying number of B cell sprays. Resultsindicate increased background with 2 sprays compared to 20 ul celldelivery. Number of sprays did not affect peak intensity with 50,000 cfuBa (n=1).

FIG. 108 is a graph. Ba Standard with 1-spray cell delivery. 50 ul of Basamples prepared in CO2 (I) media and tested with one spray of B cells.Results indicate similar backgrounds with 20 ul cell delivery and LOD of5,000 cfu. 50 and 500 cfu Ba showed 50% chance of detection (n=2).

FIG. 109 is a graph. Ba Standard: 500 cfu Ba detection with 20 ul Bcells. 50 ul of Ba samples with 500 cfu Ba was prepared in CO2 (I) mediaand tested with 20 ul B cells. Results 100% detection of 500 cfu evenwith higher background than normally seen (n=3).

FIG. 110 is a graph. Ba B cell Spray: 500 cfu Ba detection with 1-sprayB cells. 50 ul of Ba samples with 500 cfu Ba was prepared in CO2 (I)media and tested with 1 spray of B cells. Results indicate 50% detectionof 500 cfu and a 2-3× higher background (n=14).

FIG. 111 is a graph. Ba B cell Spray: 500 cfu Ba detection with 1-sprayB cells and no spin. 50 ul of Ba samples with 500 cfu Ba was prepared inCO2 (I) media and tested with 1 spray of B cells. Samples were not spunfor 5 seconds before reading. Results indicate no cell to agentinteraction resulting in 0% detection of 500 cfu Ba (n=3).

FIG. 112 is a graph. Yp B cell Spray: 500 cfu Yp detection with 20 ul Bcells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2 (I) mediaand tested with 20 ul B cells. Results indicate a typical background and100% detection of 500 cfu Yp (n=4).

FIG. 113 is a graph. Yp B cell Spray: 500 cfu Yp detection with 1-sprayB cells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2 (I)media and tested with 1 spray of B cells. Results indicate a slightlyincreased background with 100% detection of 500 cfu Yp (n=8).

FIG. 114 is a graph. Yp Standard: 500 cfu Ba detection with 20 ul Bcells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2 (I) mediaand tested with 20 ul B cells. Results 100% detection of 500 cfu with atypical background (n=7).

FIG. 115 is a graph. Yp B cell Spray: 500 cfu dried Yp detection with 20ul B cells. 5 ul of Yp samples with 500 cfu Yp was prepared in dH2O,dried overnight, and tested with 20 ul B cells. Results indicate 100%detection of 500 cfu Yp (n=10).

FIG. 116 is a graph. Yp B cell Spray: 500 cfu dried Yp detection with1-spray B cells. 5 ul of Yp samples with 500 cfu Yp was prepared indH2O, dried overnight, and tested with 1-spray B cells. Results indicatea higher background, but 100% detection of 500 cfu Yp (n=10).

FIG. 117 is a schematic representation of a B cell impaction experiment.

FIG. 118 is a schematic representation of an aspiration apparatus.

FIG. 119 is a schematic representation of aspiration based onBernoulli's principle.

FIG. 120 is a schematic representation one embodiment of the invention.

FIG. 121 is a pair of photographs illustrating a portable 16 channelsensor of the present invention. The sensor consists of a rotor holdingassay tubes horizontally, equally distributed about its circumference,and driven by a variable speed motor about a vertical axis. A singlefixed photon-detecting element, in this case a PMT, is positioned in theplane of the rotor just beyond the path of the tubes during rotation. Inthis way each of the tubes is sequentially and repetitively brought intoclose proximity to the PMT, allowing its light output to be sampled oneach pass. Finally, an optical switch consisting of an optical source(an infrared LED) and a detector (a phototransistor) is used to controlthe counting of detected photons and the reorganization of the data into16 fields, each associated with a specific sample.

FIG. 122 is a schematic of possible methods of collecting suspectedantigen onto the sample surface: (a) air impaction; (b) electrostaticcollection; (c) electrophoretic collection from a liquid sample; (d)2-part collection from a liquid sample: incubation with functionalizedmagnetic beads which capture antigen; capture of the beads by attractionin the non-uniform magnetic field of a magnetized ‘pin’ (in this case,using a sharp tip, rather than the ‘head’, may be desirable, as theforce on the magnetic beads is related to non-uniformities in themagnetic field).

FIGS. 123( a)-(c) are schematics: FIG. 123( a) a ‘straight pin’ withparticles collected on the ‘head’; FIG. 123( b) cross-section of sampletube with media and settled CANARY B-cells; FIG. 123( c) pin invertedinto B-cell tube, initiating luminous response.

FIG. 124 is a graph of pinhead dose-response curves for Bs spores driedonto pinheads and introduced (at 0 seconds) into a 200-μl tubecontaining 50 μl of settled (by centrifugation) B cells sensitive to Bs.The concentrations of the Bs spore preparation are shown in the legend.Response to a pin prepared with 107/ml of Yp is shown as a negativecontrol and an indication of a lack of cross reactivity.

FIG. 125 is a graph of Bs B-cell response to electrostatically collectedBs spores. A potential of 5.5 KV was placed across opposed pin (as inFIG. 122( b)). Concentrations of Bs spores ranging from 100-370 ACPLA(agent containing particles per liter of air) were flowed past the pinsat a rate of 2 l/min for varying lengths of time. An approximatecorrelation between collection time and B-cell response was observed.

FIG. 126 is a graph. Magnetic “Pinhead” embodiment. Bacillus subtilisparticles were dried onto the head of a pin (simulating air impaction orthe product of any other suitable localized collection method) and thenplaced into a liquid containing magnetically labeled B cells. The pinwas magnetized to attract B cells to the dried spores and placed into aluminometer.

FIGS. 127( a)-(b) are graphs of a Comparison between the standard,centrifugal CANARY assay and the dual-magnetic-bead assay. FIG. 127( a)Y. pestis in a standard CANARY assay and FIG. 127( b) Y. pestis in adual-magnetic-bead assay. Magnetic beads specific for Y. pestis weremixed with a dilution series of Y. pestis agent for 5 min. After 5 minthe magnetic beads were pulled to the bottom of the assay tube alongwith any bound Y. pestis, and the supernatant was removed. Magneticallylabeled B cells were then added to the sample and pulled down to thebottom of the tube.

FIG. 128 is a schematic. Principle of sample capture in a lateral-flowformat. Sample is added to the sample pad (˜200 μl), which in turnsaturates the pad and flows toward the capture membrane (0.2-μmmembrane). The B cells (50 μl) are then added to the sample pad andslowly wick toward the capture membrane where they encounter capturedantigen and emit photons.

FIG. 129 is a photograph of a handheld lateral-flow assay format. Asample of colored beads (1-μm diameter) were placed onto the sample padand allowed to wick up to the capture membrane to demonstrate agentcapture zone. Overall size is 1 in.×0.25 in.

FIG. 130 is a series of graphs demonstrating lateral-flow assay results:(a) E. coli in a standard CANARY assay, (b) E. coli lateral-flow assayshowing an LOD of 5000 particles per 200-ml sample, (c) B. anthracis ina standard CANARY assay, and (d) B. anthracis in a lateral-flow assayshowing an LOD of 5000 particles per 200-ml sample.

FIG. 131 is a schematic of embodiments of an automated CANARY bioaerosolsensor.

FIG. 132 is a pair of photographs illustrating an integrated CANARY diskof the present invention. The automated CANARY bioaerosol sensor diskperforms aerosol collection and CANARY B-cell storage and deliveryfunctions. For size comparison, a compact disk (CD) is shown.

FIG. 133 is a series of photographs illustrating collection details ofthe automated CANARY bioaerosol sensor disk.

FIG. 134 is a series of illustrations demonstrating the automated CANARYbioaerosol sensor disk aerosol collection optimization. A plurality ofimpactor geometries were designed and tested using CFD (ComputationalFluid Dynamics) modeling and performance was experimentally verified bycollection of aerosolized fluorescent 1 μm polystyrene spheres as modelparticles. All geometries tested demonstrated collection and thesimplest functional geometries were identified and used in furtherdevelopment. Other geometries tested showed useful properties includingparticle focusing and re-direction that may be useful in developingnovel impactor geometries capable of producing higher particle densitiesrelative to standard geometries.

FIG. 135 is a series of photographs of the automated CANARY bioaerosolsensor disk cell delivery—Viscous Plug Embodiment. In this embodiment achamber is formed with sufficient volume to contain CANARY B cells for asingle assay in a configuration that can be sealed in the plane of thedisk by a plug made of a high-viscosity, cell-compatible grease or gel(e.g. silicone grease, or petroleum jelly). The geometry of the wallswhere the viscous plugs are inserted into the disk is designed toconstrain the plug and keep it stable during transport and handling, butto release the plug when sufficient centrifugal force is applied duringa brief spin so that the cells are released to automatically move to theanalysis site. The gel can be selected to be more or less dense than theaqueous medium that contains the CANARY B cells. If its density isgreater than that of the B-cell medium (e.g. silicone grease) theviscous plug will settle to the bottom of the liquid in a defined regionof the disk away from the CANARY analysis site. If the density of thegel is less than that of the B-cell medium (e.g. silicone grease) theviscous plug will settle on the top of the CANARY reagent and can beused to form a seal above the reaction site to stabilize it for storageand transport to a laboratory for further confirmatory testing.

FIG. 136 is a schematic illustrating automated CANARY bioaerosol sensorcell delivery—Rupturable Blister Embodiment. In this embodiment CANARYB-cell storage compartments for each analysis site are built into thelid of the disk using traditional blister-packaging materials andmethods. The shape and thickness of the blisters will be designed toenable localized pressure applied to the top of the disk to rupture theside of the blisters facing the assay sites to enable the CANARY B cellsto be delivered to the assay site with a brief spin. The disk willcontain features to support the blister evenly on the sides away fromthe reaction site and focus the applied force in a specified area toprovide for reproducible liquid delivery. The blister card incorporatingthe blisters will have openings as shown to provide for proper interfacewith the manifold directing air flow through the disk for aerosolcollection, and the whole card will be sealed to the disk to provideseparation between each of the individual analysis channels.

FIG. 137 is a graph. PANTHER Disk Bacillus Subtilis Spore ImpactionResults. Bacillus subtilis particles (200 agent containing particles perliter of air, or ACPLA) were impacted into a test disk for one minute,using an aerosol chamber and Collison nebulizer. The subsequent testchannels were subjected to B cells both specific (graph) andnon-reactive (Yersinia pestis cell line) for Bacillus subtilis. Nosignal was seen for the non-reactive (Yersinia pestis) line.

FIG. 138 is a summary of the CANARY technology.

FIG. 139 is a summary of pathogen-detecting cell lines.

FIG. 140 is a summary of universal CANARY cells for detecting emergingdiseases.

FIG. 141 is a schematic of a CANARY assay for liquid and dry samples.

FIG. 142 is an example of a CANARY assay of a liquid sample.

FIG. 143 is a schematic of toxin detection using the CANARY technologydescribed herein.

FIG. 144 is an example of CANARY detection of Botulinum toxin using beadcapture as described herein.

FIG. 145 is a summary of a CANARY assay for blood-borne pathogens.

FIG. 146 is a summary of a Chlamydia study using CANARY technology.

FIG. 147 is a summary of CANARY technology applied to agriculturalpathogens.

FIG. 148 is an example of CANARY detection of plant pathogens.

FIG. 149 is a pair of photographs of a portable 16-channel CANARYsensor.

FIG. 150 is an illustration of an automated Bioaerosol CANARY (BCAN)testbed.

FIG. 151 is an illustration of pathogen analyzer for threateningenvironmental releases (PANTHER).

FIG. 152 is an illustration of an integrated PANTHER disk development.

FIG. 153 is a summary of CANARY technology.

FIG. 154 is a summary of CANARY detection of Botulinum toxin.

FIG. 155 is a summary of CANARY bioaerosol sensor performance.

FIG. 156 is a summary of virus extraction from plant tissue.

FIGS. 157A and B illustrate plant virus detection using CANARY. FIG.157A illustrates the sample preparation. FIG. 157B graphs the results.

FIG. 158 are graphs of the results of detection of BoNT/A in urine. Nosample preparation was required for detection of BoNT/A in urinesamples. Beads conjugated to 6E10-10 antibody were added directly tourine spiked with active BoNT/A, incubated for 15 minutes, and the beadswashed. Media was added to the beads, followed by CANARY cells, and thesample spun for 5 seconds. The limit of detection was 16 ng/ml, about 5fold higher than BoNT/A diluted directly into assay medium. Controlurine (0 ng/ml), which was not spiked with BoNT/A Hc prior to beadaddition, gives no signal, indicating that any nonspecific stimulatorshave been removed.

FIG. 159 are graphs of the results of detection of BoNT/A Hc in blood.CANARY is capable of detecting soluble BoNT/A Hc in blood products.BoNT/A was spiked into whole blood and the plasma prepared as describedelsewhere (see Fran's section on blood sample prep). 6E10-10 antibodycoated Protein G beads were added to plasma, incubated 2 minutes, washedinto media and assayed using 6B2-2 cells. The limit of detection forBoNT/A in this assay was 16 ng/ml.

FIG. 160 are graphs of the results of the effects of adding NaCl,Tween-20, and Triton X-100 to plasma. Addition of NaCl (finalconcentration of 0.5M) produced the most dramatic improvement in signalamplitude, from 1700 RLU to 4800 RLU. Addition of Tween was ineffective,but addition of Triton improved the signal marginally to 2700 RLU.Combining salt with the detergent improved the signal compared todetergent alone, but not to levels above that achieved with salt alone.

FIG. 161 are graphs of the results of BoNT/A (5 fmoles=800 pg) of andBoNT/A Complex (5 fmoles=5 ng) incubated for 20 minutes with 6E10-10antibody bound to streptavidin beads. 6B2-2 cells were added and lightoutput monitored. Similar responses by the CANARY assay to equimolaramounts of both preparations indicates that the complex proteins do notaffect antibody binding to BoNT/A.

FIG. 162 are graphs of the results of the detection of BoNT/A in controlmedium. Magnetic beads coated with 6E10-10 antibody were added to mediaspiked with the indicated concentrations of BoNT/A. The sample wasrotated for 2 minutes to allow toxin binding to the beads. 6B2-2 CANARYcells were added, the mixture spun for 5 seconds and light emissionmonitored. Samples containing 160 pg of BoNT/A Hc (16 ng/ml) producedsignals 6 fold over background.

FIG. 163 is a bar chart. Equal amounts of various radiological materialscovering all three emitter types (alpha, beta, and gamma) were analyzed.The response of CANARY compares favorably to a commercial,laboratory-based instrument.

FIG. 164 is a schematic of chemical detection by CANARY cells.Conceptually, detection of chemicals, such as biowarfare agents, byCANARY is very similar to detection of protein toxins. Periplasmicbinding proteins (PBPs) are attached to the surface of a bead. Thepresence of the target chemical converts the PBP into a form that isrecognized by antibodies on the surface of the CANARY cell, therebystimulating light emission.

FIG. 165 is a graph. The data points represent the average efficiencymeasured from 6 impaction runs with polydisperse polystyrene-latexspheres aerosolized with the Pitt generator. A single APS particle sizerwas switched between the inlet and the outlet of the impactor. The solidred line is an exponential fit to the averaged data between 0.7 and 1.5μm. Above 2 μm, the calculated efficiency numbers become less reliabledue to the lower aerosolization efficiency (and therefore low particlecounts) for these larger particles in the Pitt generator.

FIGS. 166A and B illustrate the method and results of dry identificationof Bacillus subtilis spores. As shown in the schematic of the dry assayprotocol (FIG. 166A), B Cells specific for B. subtilis are added and abrief centrifugal spin drives the cells to the collection site at thebottom of the sample tube. Specificity demonstrations in the dry assayformat (FIG. 166B): Blue curve: B. subtilis impacted onto sample tubesand detected with cells against B. subtilis demonstrate the ability ofCANARY to detect impacted samples. Negative Controls: baseline responseswere observed for exposure of B. subtilis cells to dried V. cholerae(Gray curve), air contaminants impacted in an empty tube in the absenceof pathogen (Red curve), and when cells against Yersinia pestis wereexposed to dry-impacted B. subtilis spores (Black curve).

FIG. 167 illustrates BCAN carrier for automated bioaerosol collectionand CANARY analysis.

FIG. 168 illustrates a CANARY Disc (CD) designed to integrate aerosolcollection and B-cell delivery.

FIG. 169 illustrates the aerosol collection module cutaway withimpaction nozzle and transparent tube.

FIG. 170 illustrates a CANARY B-cell delivery module with valve deliverysystem.

FIG. 171 illustrates a TCAN-2 automated biosensor with light-tight coverremoved.

FIG. 172 illustrates one embodiment of the invention. The PANTHER disk(left) is a self-contained bioaerosol sampling and CANARY analysis toolthat can be loaded like a CD into the portable PANTHER CUB sensor(center) or ultimately into a high-throughput automated point detectionand identification sensor (right).

FIGS. 173A and B are schematic diagrams of an embodiment of theinvention as described herein.

FIG. 174 is a further schematic of an embodiment of the invention.

FIG. 175 is a bar chart illustrating the relationship between the flowrate and the size of particles that are effectively transported throughthe disk and directed to impact onto the collection surface of thedevice described herein.

FIG. 176 illustrates an embodiment of the invention.

FIG. 177 illustrates a compact sensor equipment that automaticallyprocess the illustrated CANARY disks.

FIG. 178 illustrates the core components of a CANARY device describedherein.

FIG. 179 is a graph of the performance characteristics of the CUBsensor. Typical signals from CUB analysis of Bacillus subtilis sporeaerosols.

FIG. 180 illustrate different embodiments of the invention.

FIG. 181 is a schematic illustration of positive and negativedielectrophoresis concepts.

FIG. 182 Fabrication process for DEP test chips.

FIGS. 183A and B illustrate (a) the basic design of DEP chip, consistingof interdigitated tungsten thin film electrodes on a quartz substrate.(b) a table of combinations of linewidths and spacings.

FIG. 184 is a schematic of the principle of sample capture in alateral-flow format. Sample is added to the sample pad (˜200 μl), whichin turn saturates the pad and flows toward the capture membrane (0.2-μmmembrane). The B cells (50 μl) are then added to the sample pad andslowly wick toward the capture membrane where they encounter capturedantigen and emit photons.

FIG. 185 is a photograph of a handheld lateral-flow assay format. Asample of colored beads (1-μm diameter) were placed onto the sample padand allowed to wick up to the capture membrane to demonstrate agentcapture zone. Overall size is 1 inch×0.25 inches.

FIGS. 186A-D are graphs of lateral-flow assay results: FIG. 186A: E.coli in a standard CANARY assay, FIG. 186B: E. coli lateral-flow assayshowing an LOD of 5000 particles per 200-ml sample, FIG. 186C: B.anthracis in a standard CANARY assay, and FIG. 186D: B. anthracis in alateral-flow assay showing an LOD of 5000 particles per 200-ml sample.

FIGS. 187A and B are graphs of a cComparison between the standard,centrifugal CANARY assay and the dual-magnetic-bead assay. (a) Y. pestisin a standard CANARY assay and (b) Y. pestis in a dual-magnetic-beadassay. Magnetic beads specific for Y. pestis were mixed with a dilutionseries of Y. pestis agent for 5 min. After 5 min the magnetic beads werepulled to the bottom of the assay tube along with any bound Y. pestis,and the supernatant was removed. Magnetically labeled B cells were thenadded to the sample and pulled down to the bottom of the tube.

FIG. 188 is a schematic of a handheld sensor cartridge. (a) Cartridgewith magnetic swab in stored position. (b) Swab withdrawn fromcartridge. (c) Swab ready for use, with: 1—grip, 2—protective sleeve(withdrawn), 3—shaft, 4—magnetic tip. (d) Swab ready for insertion toread position. (e) Cartridge with swab in read position: 5—B-cellcapsule rotated into place to receive the magnetic tip, 6—photonemission through hole in bottom of cartridge.

FIG. 189 is a pair of schematics of a handheld CANARY sensor design.This design reorients the PMT so that it is directed directly at thebottom of the assay tube where the CANARY cells collect for maximumsignal collection. The sample-insertion mechanism was designed tooperate via a sliding mechanism that allowed easy operation, minimumdistance between the sample and the PMT, and shielding of the PMT fromambient light during the sample load step to minimize instrument noiseduring readout. The handle of the door was designed to incorporate astrong rare earth magnet for sample manipulation, and a tube receiver tohold the tube in position near the magnet while these manipulations areperformed.

FIG. 190 is a series of photographs of a handheld CANARY sensorprototype. The PMT has a bialkali cathode with an effective spectralsensitivity range of 300-650 nm. It is operated in photon-counting modeand the signals are recorded and can be transferred to a laptop via aRS-232, 9-pin plug for further analysis. The handheld unit has theoption of running for up to 8 h using rechargeable NiCd batteries builtinto the sensor or running indefinitely when attached to a 12-V powersupply (also used for recharging the internal battery pack

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods for detecting solubleantigens. For example, the soluble antigen can be a soluble protein or achemical. In one embodiment, the soluble antigens comprise only one ortwo antigenic epitopes. Detection of soluble antigens using an antibodyexpressed on the surface of a cell, whereby binding of the antibody tothe antigen triggers an increase in calcium concentration which in turnstimulates an emittor molecule to emit a photon in response to theincrease in intracellular calcium depends on the ability of the antigento crosslink (or aggregate, thereby immobilizing the antibody on thecell surface) the antibodies on the cell surface, thereby stimulating anincrease in intracellular calcium. A soluble antigen can be inefficientat crosslinking antibodies expressed on the surface of a cell, andtherefore is inefficient at stimulating an increase in intracellularcalcium. Described herein are methods for detecting a soluble antigenwherein crosslinking of antibodies is achieved by the methods described,which stimulate an increase in intracellular calcium and cause emissionof a photon from an emittor molecule that responds to the increase incalcium concentration.

The soluble antigens and chemicals of interest to be detected include awide variety of agents. For example, and without limitation, the methodsof the invention described herein can be used to detect protein toxinssuch as Botulinum toxins, serotypes A, B, C, D, E, F, G, Staphylococcalenterotoxin-B (SEB) and other superantigens, ricin, pertussis toxin,Shiga toxin, conotoxins, Clostridium perfringens epsilon toxins,Shiga-like ribosome inactivating proteins, other soluble bacterialproducts, such as F1 antigen from Y. pestis, protective antigen, Lethalfactor, edema factor from B. anthracis. Other molecules of interest indetecting include bacterial quorum sensing molecules, e.g., homoserinelactones. Examples of chemical warfare agents, or their breakdownproducts after hydrolysis that can be detected using the methodsdescribed herein include, without limitation, cyanide (Hydrocyanicacid), Phosgene (Carbonic dichloride), CK (Cyanogen chloride), CL(Chlorine), CX (Carbonimidic dichloride, hydroxy), DP (Carbonochloridicacid, trichloromethyl ester), GA, Tabun (Dimethylphosphoramidocyanidicacid, ethyl ester), GB, sarin 9Methylphosphonofluoridic acid,(1-methylethyl)ester), GD, Soman (Methylphosphonofluoridic acid,1,2,2-trimethylpropyl ester), GF (Methylphosphonofluoridic acid,cyclohexyl ester), Mustard (1,1′-Thiobis[2-chloroethane]), HN-1,Nitrogen Mustard (2-Chloro-N-(2-chloroethyl)-N-ethylethanamine), HN-2,Nitrogen mustard (2-Chloro-N-(2-chloroethyl)-N-methylethanamine),Lewsite ((2-Chloroethenyl)arsonous dichloride), PFIB(1,1,3,3,3-pentafluoro-2-trifluoromethyl-1-propene), Triphosgene(Carbonic acid, trichloromethyl ester), V-gas (Methylphosphonothioicacid, S[2-(diethylamino)ethyl]O-2-methylpropyl ester), VX(Methylphosphonothioic acid, S-[2-[bis(1-methylethyl)amino]ethyl]O-ethylester), binary components of VX (O-Ethyl O-2diisopropylaminoethylmethylphosphonite and Sulfur), binary components of GD (Methylphosphonyldifluoride (DF) and a mixture of pinacolyl alcohol and an amine, binarycomponents of GB (Methylphosphonyl difluoride (DF) and a mixture (OPA)of isopropyl alcohol and isopropyl amine. Additionally, otherbiologically-derived chemicals can also be detected by the methods ofthe present invention, including Mycotoxins, particularly trichothecene(T2) mycotoxins, Diacetoxyscirpenol Diverse group, Saxitoxin, or otherdinoflagellage products, Microcystins (various types), Palytoxin,Satratoxin H, Aflatoxins, and Tetrodotoxin.

Additional proteins of interest to detect include, APP (AmyloidPrecursor Protein), prion proteins associated with CJD, BSE, Scrapie,Kuru, and PSA (prostate specific antigen). Furthermore, the detection ofappropriate soluble antigens or chemicals is useful in a variety ofapplications, such as clinical applications, for example, thyroidfunction, adrenal function, bone metabolism, fertility, infertility,IVF, pregnancy, growth and growth hormone deficiency, diabetes,hematology, cardiac function, cancer, allergy, autoimmune diseases,therapeutic drug monitoring, drugs of abuse, research immunoassayapplications, genetically engineered proteins, milk drug residue, liverfunction, antibiotics and antibiotic synthesis pathways. Suitablesoluble antigens for analysis in these applications are known by thoseof skill in the art (see, for example, The Immunoassay Handbook” (secondedition), David Wild, ed. Nature Publishing Group 2001. NY N.Y.).

The present invention also provides for the detection and identificationof specific nucleic acid (NA) sequences. In one embodiment, antigens areattached to the target NA using oligonucleotide probes. These probesdecorate specific NA sequences with antigen(s). This antigen-decorated(also referred to herein as antigen-conjugated) oligonucleotide iscapable of stimulating emittor cells expressing antibody against thatantigen. Free probe, if present, is monomeric, and therefore does notstimulate emittor cells. Likewise, background binding of labeledoligonucleotide to nonspecific sites on NA will not significantlystimulate the emittor cells, because the antigens resulting from theserare background binding events will be too disperse to effectivelycrosslink antibodies.

The choice of antigen depends on many factors, including theavailability and characteristics of corresponding antibodies, theabsence of crossreactive antigens in the samples to be tested, and thesolubility, stability, and cost of the antigen-oligonucleotideconjugate, as will be understood by one of skill in the art. As usedherein, an oligonucleotide can be DNA, RNA, peptide nucleic acid (PNA),locked nucleic acids, or any variety of modified nucleic acid surrogatesthat have specialized and unique characteristics as is known in the art.Additionally, the addition of cationic amino acids (in peptide orprotein form) to such probes can increase hybridization rates. Ifdesired, those cationic peptides/proteins could serve double-duty as theantigen detected by the emittor cell. Therefore, in one embodiment ofthe invention, a detection system based on emittor cells having one ormore antibodies on their surface and comprising a compound (an emittormolecule) that emits a photon upon stimulation by antigens that aremultimeric due to the presence of target NA, in particular, photonemission is stimulated by an increase in intracellular calciumconcentration.

Also provided in the invention described herein is a sensor cell thatdetects a target particle that is bound by one or more antibodies.Specifically, the sensor cells comprise an emittor molecule and an Fcreceptor that binds to an antibody which is bound to the target agent orparticle. In one embodiment, the sensor cell comprising an Fc receptoris a macrophage cell, such as the human macrophage cell line U937. Othersuitable cells or cell lines will be known to those of skill in the art.The Fc receptors are a family of membrane-expressed proteins that bindto antibodies or immune complexes. They are expressed on severalhematopoietic cells including monocytes and macrophages. Severalsubclasses of Fc receptors exist including Fc gamma Receptor I (FcγRI),a high-affinity binder of soluble antibody. FcγRI binds to the constantregion (Fc portion) of Immunoglobulin G (IgG) leaving theantigen-binding region of the antibody free. Crosslinking of theantibody-bound Fc receptor by specific antigen initiates a signalingpathway that stimulates calcium release. Therefore, crosslinking of theFc receptor on the sensor cell results in an increase in intracellularcalcium concentration and the emittor molecule thereby emits a photon inresponse to the increase in calcium concentration.

Also provided in the invention described herein is a 16-Channel Sensor.In its simplest form, an emittor cell assay consists of preparing asample in a transparent tube, introducing an aliquot of speciallyprepared emittor cells into the tube, driving the emittor cells to thebottom of the tube using a quick centrifugal spin, and measuring thelight output from the tube with a photon-counting sensor. In thelaboratory, most emittor cell assays are made sequentially, one sampleat a time; in the automated BAWS/CANARY instrument, four samples aremeasured simultaneously, each sample having its own light-gatheringchannel. The former system requires more time, while the latter requiresmore complex (and expensive) hardware.

A different approach that reduces the time to measure multiple samples(while keeping the hardware requirements minimal) is described herein. Asensor has been designed that allows the simultaneous measurement of aplurality of samples using a single light-gathering channel. The sensorconsists of a rotor holding sixteen 1.5-ml tubes horizontally, equallydistributed about its circumference, and driven by a variable speedmotor about a vertical axis (FIG. 39). A single fixed photon-detectingelement (for example, a PMT) is positioned in the plane of the rotorjust beyond the path of the tubes during rotation. In this design, eachof the tubes is sequentially and repetitively brought into closeproximity to the photon-detecting element, allowing its light output tobe sampled on each pass. Finally, an optical switch consisting of anoptical source (an infrared LED) and a detector (a phototransistor) isused to control the counting of detected photons and the reorganizationof the data into the 16 fields, each associated with a specific sample.

A further implementation of this 16-channel design is referred to as aTCAN sensor. The TCAN (Triggered-CANARY) biosensor is an automatedbiosensor which combines both aerosol collection and emittor cell liquiddelivery into an integrated radial disc format. The TCAN CANARY disc(CD) (FIG. 42) interfaces with a manifold assembly which splits an airflow into separate channels. The aerosol collection assembly (FIG. 43)uses dry impaction techniques to then localize particles from the airflow into the bottom of clear plastic tubes.

After impaction of aerosol particles, the CD interfaces with themanifold assembly to actuate valves located in the disc. The disc israpidly spun, which in turn causes the emittor cell liquid to deliver toindividual tubes using centrifugal force (FIG. 44). An optical detectoris then used to identify potential bioagents based on the photon outputof emittor cells interacting with the aerosol particles. This process ofaerosol collection and emittor cell delivery can be repeated severaltimes in one disc. This feature allows multiple emittor cell assays tobe performed after several trigger events without changing the CD.

The materials and procedures suitable for use in the invention aredescribed in further detail below.

Emittor Cells

The emittor cell (also referred to herein as a sensor cell or a CANARYcell) can be any prokaryotic or eukaryotic cell that has a suitablereceptor, signaling pathway, and signal output method, either naturally,through genetic engineering, or through chemical addition. The cell canbe an artificial or nonliving unit provided that it has a functionalreceptor, signaling pathway, and signal output method. Upon binding ofantigen receptor, such as to the antibodies, the cell mobilizes calciumions into the cytosol. An example of a cell useful in the device andmethods of the invention is a B cell (i.e., a B cell from a cold orwarm-blooded vertebrate having a bony jaw) which can be geneticallyengineered to express one or more surface-bound monoclonal antibodies.Another example of a cell useful in the device is a macrophage cell,such as the human cell line U937, which expresses an Fc receptor on thecell surface. An antigen can be bound to an antibody by addition of theantibody to the target and this antigen-antibody complex will bind tothe Fc receptor on the cell and stimulate signaling which results in anincrease in intracellular calcium.

A monoclonal antibody can be produced by, for example, immunizing ananimal with the antigen to be detected and harvesting the B cell fromthe immunized animal. DNA encoding the monoclonal antibody can then beisolated and transferred into an immortalized cell line and the cellsscreened for production of a surface monoclonal antibody specific forthe antigen to be detected. B cells are useful for both qualitative andquantitative analyses, particularly because the emission signal fromthem typically does not significantly diminish as additional targetspecimen is exposed to it and also because such emission signal islinear.

Alternatively, the cell can be a fibroblast. However, fibroblasts do notcontain the signal transduction machinery necessary to transfer a signalfrom the cytoplasmic portion of a surface antibody to calcium stores inthe cell. To overcome this problem, a chimeric surface antibody can beexpressed in the fibroblast. This chimeric antibody contains acytoplasmic amino acid sequence derived from a polypeptide (e.g., afibroblast growth factor receptor) that can transduce a signal from theinner surface of the plasma membrane of the fibroblast to intracellularcalcium stores. Thus, when an antigen binds to the extracellular portionof the chimeric antibody to cause antibody aggregation on the surface,calcium mobilization is induced. A similar strategy using chimericantibodies can be employed for any other cell type which is not a Bcell, so that the cell is suitable for use in the devices and methods ofthe invention.

Cells useful in the devices and methods herein are those designed torecognize a specific substance, including those having receptors ontheir surface that specifically bind to that substance. A preferredreceptor is an antibody or single-chain antibody, although othersuitable receptors include a mitogen receptor (such as alipopolysaccharide (LPS) receptor), a macrophage scavenger receptor, a Tcell receptor, a cell adhesion molecule, a DNA binding protein such aspart of a sequence-specific restriction enzyme or transcription factor,single-stranded-RNA- or double-stranded-RNA-binding protein, anoligonucleotide complementary to a DNA or RNA sequence to be recognized,or other ligand-binding receptor (e.g., Fas; cytokine, interleukin, orhormone receptors; neurotransmitter receptors; odorant receptors;chemoattractant receptors, etc.) that will specifically bind thesubstance to be recognized. The receptor can be attached to the cellsurface via a transmembrane domain, a membrane-bound molecule thatspecifically binds to the receptor (such as Fc receptors bind toantibodies), or a covalent or noncovalent attachment (e.g.,biotin-streptavidin, disulfide bonds, etc.) to a membrane-boundmolecule. The receptor can also be a chimeric molecule; for instance, itcan have an extracellular domain such as an antibody, single-chainantibody, lectin or other substance-specific binding domain or peptide,and an intracellular domain such as that from the insulin receptor,fibroblast growth factor, other protein that triggers a second messengercascade, etc. Instead of directly binding to the substance to berecognized, the receptor might specifically bind to another molecule orobject that in turn specifically binds to the substance to berecognized, such as a secondary antibody, labelled bead,antigen-conjugated oligonucleotide, etc.

Alternatively, only one of these binding steps may need to be specific.For instance, DNA or RNA containing specific sequences may be pulled outof solution using oligonucleotide probes conjugated to one antigen (ordirectly to a bead, or on a matrix), and a second set of nonspecificantigen-conjugated oligonucleotide probes annealed to the target DNA/RNAwould be used to stimulate cells specific for that second antigen. Also,non-specific nucleic acid binding proteins (histones, protamines,RNA-binding proteins) expressed as chimeras on the cell surface, orantibodies against those binding proteins, could also be used to detectthe presence of nucleic acids after a sequence specific selection step.

Antibodies

Whatever original cell type, the antigen-binding variable regions ofmonoclonal antibodies can obtained either as DNA sequence from a publicsource, or cloned by RT-PCR from a hybridoma cell line. RT-PCR isaccomplished using sets of primers designed to anneal, at the 5-primeend, to either the leader or framework regions of the variable region,and at the 3-prime end to the constant region.

The antibody variable regions are then cloned into expression vectorsthat already contain the constant regions for light and heavy chain. Thelight chain expression vector described in Persic et al., Gene 187:9-18,1997 is especially suitable for this purpose. VKExpress, described inPersic et al., contains the EF-1α promoter, a leader sequence, multiplecloning sites, and the human Ig kappa constant region andpolyadenylation signal. The heavy chain expression vector is derivedfrom Invitrogen's pDisplay. This vector contains a CMV promoter, aleader sequence, an HA tag, multiple cloning site, and myc tag, followedby the PDGFR transmembrane domain and bovine growth hormonepolyadenylation signal.

pDisplay can be modified for heavy chain expression as follows. ThePDGFR transmembrane domain of pDisplay is replaced with the murine IgMconstant region without the exon that allows for secretion. This ensuresthat the protein will remain membrane-bound. The neomycin-resistancegene can be replaced by any of a number of antibiotic-resistance genesincluding, but not limited to, hygromycin, bleomycin, puromycin,kanamycin, and blasticidin genes. The heavy chain (or alternativelylight chain) variable region can be inserted in a two-step process,using overlap-extension PCR, to remove the HA and myc tags present oneither side of the multiple cloning site of pDisplay. A vector can alsobe developed to allow insertion of an overlap extension productcontaining the variable region fused to approximately 300 base pairs ofthe IgM constant region, so that cloning can be done in a single step.

The examples below were implemented using the antibody vectorconstruction procedure described immediately above.

An antibody which specifically binds to the antigen to be detected is amolecule which binds to the antigen or an epitope of the antigen, butdoes not substantially bind other antigens or epitopes in the sample.Such antibodies can be chimeric (i.e., contain non-antibody amino acidsequences) or single chain (i.e., the complementarity determining regionof the antibody is formed by one continuous polypeptide sequence).

Alternatively, surface antibody-producing cells can be obtained from theanimal and used to prepare a monoclonal population of cells producingsurface antibodies by standard techniques, such as the hybridomatechnique originally described by Kohler et al., Nature 256:495-497(1975); Kozbor et al., Immunol Today 4:72 (1983); or Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., pp. 77-96(1985). The technology for producing cells expressing monoclonalantibodies is well known (see, e.g., Current Protocols in Immunology(1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.),with modifications necessary to select for surface antibodies ratherthan secreted antibodies.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating acell producing a surface monoclonal antibody (see, e.g., CurrentProtocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977;Kenneth, In Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner,Yale J Biol Med 54:387-402 (1981). Moreover, the ordinarily skilledworker will appreciate that there are many variations of such methodswhich also would be useful.

Polyclonal cells expressing antibodies can be prepared by immunizing asuitable animal with the antigen to be detected. The cells producingantibody molecules directed against the antigen can be isolated from theanimal (e.g., from the blood) and further purified by well-knowntechniques, such as panning against an antigen-coated petri dish. As analternative to preparing monoclonal cells, a nucleic acid encoding amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the antigen to thereby isolateimmunoglobulin library members that bind the antigen. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal., Bio/Technology 9:1370-1372 (1991); Hay et al., Human AntibodHybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989);Griffiths et al., EMBO J. 12:725-734 (1993).

After the desired member of the library is identified, the specificsequence can be cloned into any suitable nucleic acid expressor (e.g., avector) and transfected into a cell such as a fibroblast. The expressorcan also encode amino acids operably linked to the antibody sequence asappropriate for the cell which is to express the antibody. As discussedabove, the cytoplasmic transmembrane sequence of a fibroblast growthfactor receptor can be linked to a single-chain antibody specific forthe antigen to be detected, so that the cell immobilizes calcium whencontacted with the antigen. Although separate recombinant heavy chainsand light chains can be expressed in the fibroblasts to form thechimeric antibody, single chain antibodies also are suitable (see, e.g.,Bird et al., Trends Biotechnol 9:132-137, 1991; and Huston et al., IntRev Immunol 10:195-217, 1993).

Photon Emitter Molecules

Binding of the desired substance to the cell-surface receptor shouldtrigger a signaling pathway inside the cell. A preferred signalingpathway is the second-messenger cascade found in B cells, T cells, mastcells, macrophages, and other immune cells, wherein crosslinking of thecell surface receptors activates a tyrosine kinase, which thenphosphorylates phospholipase C, which then cleaves phosphatidylinositol4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol; IP3 then opens calcium channels to release calcium fromintracellular stores such as the endoplasmic reticulum or to let inextracellular calcium, thereby elevating the calcium concentration inthe cell's cytosol. Depending on the receptor type, cell type, anddesired signaling method, alternative second-messenger cascades could beemployed, such as a G-protein-adenylyl cyclic-cAMP-protein kinase Acascade.

A method should be provided for monitoring the internal signaling of thecell in response to substances to be identified. If the internalsignaling involves an increase in cytoplasmic calcium, a preferreddetection method is a calcium-sensitive luminescent or fluorescentmolecule, such as aequorin, obelin, thalassicolin, mitrocomin(halistaurin), clytin (phialidin), mnemopsin, berovin, Indo-1, Fura-2,Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, chameleons (A. Miyawaki etal., (1999) Proc. Natl. Acad. Sci. 96, 213540), or similar molecules. Itis anticipated that the relative intensities of light and the sensorcell storage characteristics enabled by using calcium-sensitivemolecules may vary depending on the efficiency of light production forthe specific emitter molecule and the half-life of the activated emittermolecule—in some cases providing significant benefits (e.g., improvedsensitivity, quantitative or qualitative detection). Additionalperformance enhancements may arise from the use of structural analogs ofthe natural cofactors of photoprotein emitter molecules. Variouscalcium-sensitive fluorescent dyes which can be taken up by live cellsare available from commercial sources, including Molecular Probes, Inc.,Eugene, Oreg. Proteins such as aequorin, obelin, thalassicolin,mitrocomin (halistaurin), clytin (phialidin), mnemopsin, berovin orchameleons could be added genetically, injected into the cells, ordelivered by a protein uptake tag from HIV TAT (approximately aminoacids 47-57; A. Ho et al. (2001) Cancer Research 61, 474-477) or byother means. If desired, such reporter molecules can include targetingsignals to target them to the cytoplasmic face of the endoplasmicreticulum or the plasma membrane, the interior of the mitochondria, orother locations where the change in local calcium concentration might beparticularly large. Optical methods of detecting activity from otherpoints in the signaling pathway could also be used, such as fluorescenceresonance energy transfer (FRET) of fluorescent groups attached tocomponents of the signaling pathway (S. R. Adams et al. (1991) Nature349, 694-697). Where the internal signaling involves an increase inreactive oxygen species (e.g. superoxide anion radicals, hydroxylradicals, compound I or II of horseradish peroxidase, etc.), a preferreddetection method is a reactive-oxygen-sensitive luminescent orfluorescent molecule, such as the photoprotein pholasin (a 34-kDaglycoprotein from the bioluminescent mollusc, Pholas dactylus) orsimilar molecules. Alternatively, a reporter gene for any luciferasecould be linked to a promoter induced by the signaling pathway. In somecells such as T cells and mast cells, the signaling pathway triggersexocytosis of granules containing proteases such as granzymes,tryptases, or chymases. Exocytosis of these proteases could be detectedby calorimetric or fluorometric methods (e.g., p-nitroanaline or7-amino-4-trifluoromethyl coumarin (AFC) linked to peptides cleaved bythe proteases [S. E. Lavens et al. (1993) J. Immunol. Methods 166, 93;D. Masson et al. (1986) FEBS Letters 208, 84; R&D Systems]). Also,microelectrodes or other methods to detect the electrical activityassociated with the calcium flux or other signaling ion fluxes aresuitable to monitor signaling response in the cell.

A suitable emitter molecule is any molecule that will emit a photon inresponse to elevated cytosolic calcium concentrations, includingbioluminescent and fluorescent molecules. One emitter molecule, thebioluminescent aequorin protein, is described in Button et al., CellCalcium 14:663-671 (1993); Shimomura et al., Cell Calcium 14:373-378(1993); and Shimomura, Nature 227:1356-1357 (1970). Aequorin generatesphotons by oxidizing coelenterazine, a small chemical molecule.Coelenterazine diffuses through cellular membranes, so coelenterazine oran analog thereof can be added to the culture medium surrounding thecells. Alternatively, genes encoding enzymes that make coelenterazinecan be introduced into the cells. In another embodiment, bioluminescentgreen fluorescent protein (GFP) (see Chalfie, Photochem Photobiol62:651-656 [1995]) or yellow fluorescent protein (YFP) can be used. Inthis embodiment, the cell cytosol contains both GFP and aequorin. Inresponse to elevated calcium in the cytosol, aequorin donates energy toGFP in an emissionless energy transfer process. GFP then emits thephoton. Alternatively, the emitter molecule can be a calcium-sensitivefluorescent molecule (e.g., indo-1) which is illuminated by a wavelengthof light suitable to induce fluorescence.

Aequorin, or any other emitter molecule, can be introduced into the cellby methods well known in the art. If the emitter molecule is a protein(as is the case with aequorin), the cell can contain an expressionvector encoding the protein (i.e., a nucleic acid or virus which willproduce the emitter molecule when introduced into a cell). An expressionvector can exist extrachromosomally or be integrated into the cellgenome.

Conjugated Antigens/Tags

One or more antigens or tags can be added (also referred to herein asconjugated) to molecules to provide a known antigenic epitope. Forexample, one or more antigens can be conjugated to an oligonucleotide toproduce an antigen-conjugated oligonucleotide with a known antigenicepitope. An antigen-conjugated molecule can comprise one antigen ormultiple antigens that are either the same of different. For example andwithout limitation, an antigen or tag to be conjugated to a molecule fordetection includes small antigens such as digoxigenin, digoxin,phosphocholine, fluoroscein or other fluorphores, and biotin, andpeptides such as HIS, VSV-G, FLAG, and C(AAKK) multimer (as described inCorey, J. Am. Chem. Soc., (1995) 117: 9373-4).

Oligonucleotides

In addition to conventional DNA and RNA probes, a variety of modifiednucleic acids have been shown to hybridize in a sequence-specific mannerto target nucleic acid sequences. These include peptide nucleic acids(PNA) (Nielsen et al., (1991) Science 254: 1497-1500), Bis-PNAs(Griffith et al., (1995) J. Am. Chem. Soc 117: 831-832), Tail-clamp PNA(Bentin (2003) Biochemistry 42: 13987-13995), PD loops (Bukanov et al.,(1998) PNAS 95: 5516-5520), PNAs incorporating pseudocomplementary bases(Lohse et al., (1999) PNAS 96 (21) 11804-11808), or locked nucleic acids(Braasch and Corey (2001) Chem. Biol. 8: 1-7). A variety of thesemodified nucleic acids have been shown to have differ in hybridizationcharacteristics, stability, affinity, and specificity, and could be usedin place of conventional DNA oligonucleotides (reviewed by Beck andNielsen, pp. 91-114, in Artificial DNA: Methods and Applications. CRCPress, Y. E. Khudyakov and H. A. Fields eds.). Attachment of cationicproteins, peptides, or DNA binding proteins has been shown to improvehybridization kinetics (Corey (1995) J. Am. Chem. Soc 117: 9373-9374;Zhang et al., (2000) Nuc. Ac. Res. 27 (17) 3332-3338).

The binding of oligonucleotides has been shown to improve with theaddition of helper oligonucleotides (O'Meara et al., (1998) Anal.Biochem. 225: 195-203; Barken et al, Biotechniques (2004) 36: 124-132).Specificity can be improved by addition of unlabeled hairpin competitorprobes (Huang et al., (2002) Nucleic Ac. Res. 30: (12) e55).

Removal of unbound oligonucleotides after hybridization to target is notnecessary for nucleic acid sequence detection, but may be desirable. Theunbound labeled oligonucleotide could be removed using a variety ofconventional chromatography techniques, including size exclusion,hydrophobic interaction, or ion exchange, depending on the chemistry ofthe particular probe used.

Other Nucleic Acid-Binding Molecules

Oligonucleotides are not the only molecules that are able to identifyspecific nucleic acid sequences. Proteins are also capable of suchdiscrimination, and can be expressed on the surface of the emittor cell,recombinantly attached to a cytoplasmic domain that would, upon binding,initiate a calcium response. This would include nucleic acid bindingproteins attached to the Fc portion of antibodies, for example.Expression of nucleic acid binding proteins on the surface of theemittor cell would eliminate having to denature double-stranded nucleicacid prior oligonucleotide hybridization, and additionally, the systemproduces all the necessary components: no exogenously synthesizedoligonucleotides would be required. Possible sequence specific DNAbinding proteins include: (1) DNA restriction enzymes (preferably withthe DNA-cutting catalytic site removed or inactivated, e.g. L. F. Dorner& I. Schildkraut (1994) Nucl. Acids Res. 22, 1068-1074); (2)Transcription factors or other specific DNA- or RNA-binding proteins,especially those that recognize unique DNA or RNA sequences in pathogensor organisms of interest (e.g., HIV TAT transcription factor: C. Brigatiet al. (2003) FEMS Microbiology Letters 220, 57-65; poxvirustranscription factors: S. S. Broyles (2003) Journal of General Virology84, 2293-2303). Emittor cells with such receptors could be designed tocrosslink on target DNA/RNA with either a specific repeated sequence oralternatively two or more unique sequences.

Capture Oligonucleotides

Although not necessary for detection, capture of the target nucleic acidsequence on sedimentable or solid support can improve assay sensitivity.Single-stranded DNA target can be captured using, for example,biotin-labeled capture oligonucleotides bound to streptavidin-coatedpolystyrene or paramagnetic beads. The captured material can beseparated from unbound material by centrifugation or exposure to amagnetic field, as appropriate. The use of an intermediate bindingreaction (avidin-biotin) in attaching the oligonucleotide to the beadmay not be necessary as any interaction that would attach theoligonucleotide to a solid support can be used, including directconjugation. In addition, any solid support to which the captureoligonucleotide can be attached would suffice. This can be in the formof a two-dimensional array, in which specific capture oligonucleotidesare placed in specific positions on the array. Alternatively, targetnucleic acid sequences can be captured in a non-specific manner (e.g.ion exchange resin, precipitation, histone or protamine binding). Targetcapture will also concentrate the target nucleic acid sequence and/orremove assay interferents.

Polyvalence

Emittor cell stimulation is dependent on the antigen appearingmultivalent to the emittor cell. In general, this can be accomplished inat least two ways. First, multiple copies of antigen can be attached toa target molecule, for example, hybridizing multiple antigen-conjugatedoligonucleotides to the target nucleic acid sequence. Second, severalcopies of the target nucleic acid sequence, each with a single antigenattached, can be bound to each other or bound in close proximity to eachother (e.g., attached to a bead). In this example, the individual targetnucleic acid sequence would not be polyvalent, but the bead withmultiple copies of the target nucleic acid sequence attached wouldpresent a polyvalent antigen.

Reaction Chambers

The reaction chambers suitable for use in the invention can be anysubstrate or vessel to which emitter cells and candidate particles canbe mixed and contacted to each other. In one embodiment, the reactionvessel is a centrifuge tube (e.g., a microcentrifuge or Eppendorf tube).As described herein, centrifugation is a particularly well-suited meansto pellet candidate particles or emitter cells first, before the otheris driven into the first pellet. To further increase the pelleting ofboth particles and cells, the side walls of the tube can be coated witha non-sticky carrier protein such as bovine serum albumin to prevent thesticking of emitter cells to the side walls, and the bottom of the tubecan be coated with poly-L-lysine to help ensure that the targetparticles stay adhered to the bottom of the tube. Other proteins ormolecules that either prevent or promote cell adhesion are known in theart of cell biology and are suitable for use in the invention.

Centrifuge tubes with customized sample well geometries can provide anadditional embodiment that uses centrifugation to increase emittor cellinteractions with difficult-to-sediment particles and reduces the needto customize spin sequence. In this embodiment the particle-containingsample to be analyzed is placed in a tube where the maximum width of thesample chamber is approximately equal to the diameter of an emittercell. Layering a concentrated emitter cell suspension over the samplefollowed by centrifuging drives a large number of closely packed emittercells through the smaller particles while the constrained geometryincreases the probability of emitter cell antibody interaction withparticles. Binding of the cell-associated antibody to the particlecaptures the poorly sedimenting particle and will rapidly draw it to thebottom of the tube with the emitter cell where the resulting light canbe observed by a photo multiplier device.

In another embodiment, the reaction chambers are wells in atwo-dimensional array, e.g., a microtiter plate, or spots or wells alonga tape, as shown in the figures. These arrangements allow multiplexdetection of either multiple samples and/or multiple target particles.For automated delivery of candidate particles and/or emitter cells,either the reaction chambers or the specimen collector and emitter cellreservoir is addressable in at least two dimensions. The wells of arrayscan also be treated with sticky and non-sticky coatings as describedabove for centrifuge tubes to facilitate contact between emitter cellsand candidate particles.

Specimen Collectors

Different devices can be used to collect samples from, e.g., air. Ingeneral, an air sampling device has a collection chamber containingliquid through or beside which air or gas is passed through, orcontaining a porous filter that traps particulates (e.g., targetparticles) as air or gas passes through the filter. For collectionchambers containing liquid, the collection liquid can be centrifuged orotherwise treated to separate particles from the liquid. The separatedparticles are then deposited in a reaction chamber. For collectionchambers containing a filter (e.g., nitrocellulose), the filter orportions of the filter can act as the reaction chamber. Alternatively,particles can be washed from the filter, or the filter can be dissolvedor otherwise removed from the particles. A filter collection chamber canalso be adapted to collect particles from a liquid (e.g., water supplysample or cerebral spinal fluid) flowing through the filter. Inaddition, as discussed above, a liquid sample can be centrifuged toremove any particulate material present in the liquid. A variety ofsamplers are known and available for use with the present invention. SeeSKC, Inc., which sells the SKC BioSampler®. and other sampling devices.

Other air samplers can be used. For example, an alternative device isthe Air-O-Cell sampling cassette (SKC, Inc.). In this device, theairborne particles are accelerated and made to collide with a tackyslide which is directly suitable for various staining procedures andmicroscopic examination.

Aerosol particulates may be collected using inertial separation in adevice known as an impactor. An airflow containing particles to becollected is drawn from the environment of interest into the impactorwhere it is directed towards a surface for impaction. With appropriategeometrical parameters and flow rates in the impactor, particles withsufficient inertia will not follow the flow streamlines, but will impactonto the surface. A significant proportion of the particles impactingthe surface adhere through electrostatic and/or van der Waalsinteractions and are thereby collected and concentrated. In this way,aerosol particles containing proteins (including toxins), viruses,bacteria (vegetative and spore forms), parasites, pollen and otherdetectable substances can be collected for detection using a variety ofavailable assay technologies including the devices and methods herein.

Dry sample collection for bioassays using an air impactor providesgeneral advantages over traditional air-to-liquid sample collection byreducing or eliminating fluid consumables and transfer mechanisms whichreduces assay cost and simplifies automation. Of particular benefit tothe devices and methods herein, collection using dry impaction ensuresthat all of the collected sample is located on the surface prior to theaddition of sensor cells of the devices and methods herein, regardlessof the size of the individual analyte particles. This achieveslocalization of all analytes regardless of their sedimentationcoefficient in fluid, thereby maximizing the sensitivity of the devicesand methods herein and accelerating many implementations of the assay byeliminating a time-consuming step.

Any surface that retains a proportion of particles that impact onto itand that is compatible with subsequent bioassays is suitable as acollection surface. Suitable materials include biocompatible metals,plastics, glasses, crystals, aerogels, hydrogels, papers, etc.Particularly useful configurations of these materials includemicrocentrifuge tubes, multi-well plates used in high-throughputscreening, continuous tapes, filters, conjugate release pads of lateralflow immunoassays, etc. The collection efficiency can be increased bymodifications to the collection surface including: the addition ofcoatings promoting adhesion of biological particles (these coatings canbe chemical or biochemical in nature, e.g. polylysine), increasedsurface roughness to increase the surface area available for collection,and customized surface geometries that promote deposition of particlesin defined regions on the surface. Furthermore, additional improvementsin collection efficiency can be achieved by manipulating theelectrostatic charges on the collection surface and the incomingparticles such that additional attractive forces are generated.

Additional improvements can be made to the dry impaction collector byusing an air-to-air concentrator upstream of the collector to increasethe number of particles in each unit of air sample impacted onto thecollection surface. This can significantly reduce the amount of timeneeded to collect a sufficient number of aerosol particles to providereliable results for the detector.

In one example of this collection concept, the impactor described inFIG. 23 has been configured to collect aerosol samples on the bottom ofa commercially available plastic tube. A nozzle projects down into thetube and the exit is positioned at the radius of curvature of the tube'sinner surface. This positioning increases the likelihood of particleimpaction upon the tube bottom where the device sensor cells are mostlikely to contact them. Once collection is completed, a single dropletcontaining device sensor cells is added directly to the tube containingcollected aerosol particles, spun for 5 seconds to accelerate celldelivery to the tube surface, and emitted light is measured using aphoton detector (e.g., PMT, CCD, photodiode, etc.). Using thisapparatus, dry bacterial spores can be collected from an aerosol andidentified directly with optoelectronic device in less than one minute.This method can be implemented with a plurality of tubes used to collectsamples and an automated system to conduct subsequent assays. An exampleof how a system capable of conducting at least 10 independent assays isshown in FIGS. 4, 6, 9, 12, and 15. By implementing an approach whereassays are made capable of looking for multiple analytes in a singletube (multiplexed) the number of detectable substances for a singleassay cycle can be made greater than the number of available tubes. Thiscan be done by creating individual optoelectronic detection device celllines expressing a plurality of receptors with affinity for differentanalytes or by combining multiple cell lines with differentspecificities in a single tube.

FIG. 4 is a schematic of an integrated biological aerosol warning sensor(BAWS)/optoelectronic sensor system. The BAWS trigger module is used topreliminarily detect the presence of particles, e.g., those of apre-determined size range. If particles meeting specifications aredetected, BAWS triggers an air-to-air concentrator that allows particlesof a particular size range to be collected and deposited in a well(e.g., reaction chamber, tube) via a dry-impactor module. Thedry-impactor module allows for dry sample collection and is incommunication with a syringe module for cell (e.g., emitting cells)delivery into a reaction chamber (e.g., tube). A transport module isused to transfer the reaction chamber assembly (having one or morechambers or tubes) to a centrifuge module for sedimentation or mixing ofthe particle sample and cells. The centrifuge module can be, but neednot necessarily be, in communication with an optics/PMT module fordetection of photon emission. A controller module is useful for controlof operation of the system.

FIG. 6 shows an example of a dry-impactor module concept. In thisexample a single (e.g., prototype system) as well as a multi-channeldevice is illustrated, including individual sample tubes (e.g., PCRtubes) and tube carriers, in communication with air-to air concentratorsfrom which the particle test sample is collected.

FIG. 9 shows an example of a cell-delivery that can be automated. Thesensor cells (e.g., emitting cells) are introduced to the system bymeans of a syringe and syringe pump arrangement, which can includepipettors or other delivery equipment. This type of assembly allows formultiple and simultaneous introduction of sensor cells to the particlesamples (e.g., samples in reaction chambers (e.g., tubes).

FIG. 12 shows an example of a centrifuge module concept used to spin theparticle samples or cell samples. Carriers having the sample tubes areintroduced via a loading mechanism into a rotor assembly that issuitable for receiving the carriers. The rotor spins the samples. Therotor assembly is in communication with optics modules for signalcollection (e.g., photon emission), and an indexed motor can be used toallow for alignment of the samples chambers with the detector (e.g.,optics modules).

FIG. 15 shows an example of an optics module. Depending on the preciseconfiguration, the module allows for a plurality of simultaneous testingof samples (e.g., in the reaction chambers, tubes). The carrier andtubes therein are introduced to the unit such that they are incommunication with lens assemblies (e.g., integrated reflectors, lenses)if necessary, and ultimately a photodetector (e.g., a PMT). The PMTproduces signals that are then sent to a processor for processing anddisplay.

FIG. 21 illustrates an integrated dry-impactor/optoelectronic sensor. Inthis sensor the modules described above are assembled in a lineararrangement with a cassette holding 30 carriers deliverable to abelt-driven carrier transport module. This transport module moves theassay tubes sequentially from the collector to the cell delivery moduleto the centrifuge module, and finally to the confirmatory sample storagemodule following completion of photon detection. The overall size ofthis integrated sensor is approximately 54 inches wide by 33 inches highby 22 inches deep.

Real-world samples may contain substances that either inhibit the assay(false negative) or cause a response in the absence of specific antigen(false positive). In many instances, these samples can be treated priorto the assay to remove these substances. For example, soluble substancessuch as detergents or serum factors can be removed by pre-centrifugationstep, where the agent is concentrated in the bottom of the tube and theliquid is replaced with assay medium (Portal Shield samples). Insoluble,large particulate substances can be removed from the sample byfiltration, using commercial filters of a pore size (3-5 μm) that allowsthe passage of the agent, but retains the contaminant (diesel or sootsamples). Samples can be processed rapidly through syringe filters,adding only a few minutes to the total assay time.

Specimen Localization

As part of the specimen collector or reaction chamber, differentmechanisms (other than centrifugation) can be implemented to facilitatecontact between emitter cells and candidate particles. For example, theuse of electrophoresis, isoelectric focusing, dielectrophoresis,magnetically tagged particles, and the like in bioelectronic devices canbe integrated into a system of the invention. See, e.g., U.S. Pat. No.6,017,696 and other patents assigned to Nanogen, Inc.; Goater et al.,Parasitology 117:S177-189, 1998; and U.S. Pat. Nos. 5,512,439 and4,910,148 and other patents assigned to Dynal AS.

Mixing a aqueous sample containing target particles (particles here canbe anything recognized by the emitter cells-proteins/toxins, viruses,bacteria, parasites, nucleic acids, etc.) with an aliquot of mediacontaining emitter cells results in particle-cell contact leading totransient increase in the rate of photon emission. The time between thestart of the mixing process and the maximum emission rate depends on thecharacteristic response of the particular cells to stimulation as wellas the time over which the mixing occurs (the mixing time) and thetypical time for the particles and cells to come into contact aftermixing (the diffusion time).

Because a background rate of detected photons will exist even in theabsence of target particles (background cell emission and thermal noisein the photon detector and its electronics, for example), photonsemitted from single target-cell interactions can be difficult todistinguish from this background. To be useful as a signal, there mustbe a significant increase in the rate of photons detected over that ofthe background. For a given sample, this rate is maximized when themixing time and diffusion time are minimized. Other possible signalsthat target particle are present in a sample include: an increase in thetotal number of photons detected in a period of time above that of thebackground alone, a change in the statistics of detected photons, or achange in the spectral qualities of the detected photons.

The diffusion time can be minimized by reducing the average distancebetween particle and cell after mixing. This can be accomplished bylocalizing the particles and/or cells to within a small volume, often alayer, within the larger mixed volume. However, the time to localize theparticles and/or cells may be longer than the characteristic responsetime of the cells. Mixing between particles and cells over thisprolonged localization could produce a lower rate of photon emission,and therefore a lower signal, by increasing the average time betweenemissions. To avoid this, one or both should be localized separately,while minimizing contact between them. This localization can also leadto a reduced mixing time.

Generally, the means to move particles or cells include the following:sedimentation (by gravity or centrifuge); fluid flow (forced orconvective); electric forces (electrophoresis and dielectrophoresis);magnetic forces (using magnetic beads); and acoustics/ultrasonics(standing or traveling waves).

Localization requires a means of moving particles and/or cells combinedwith a barrier where particles and/or cells can collect, such as thesolid surface of a channel or container, the surface of a filter, or thepotential energy barrier surrounding an electric-field minimum. Examplesinclude: sedimentation (localizing cells on the lower surface of achamber); air impaction (impacted particles stick to or settle onto acollection surface); filtering (particles or cells collect on to thesurface or into the body of a filter); affinity capture particles orcells can be localized through specific or non-specific bindinginteractions); magnetic capture (magnetic beads held against a solidsurface, a filter surface, or in the body of a filter by localizedmagnetic forces; beads may or may not have surface chemistry to promoteattachment of particles or cells); electrophoresis (charged particlesonly; collection on to an electrode surface); and dielectrophoresis(positive: collection of particles or cells on to an electrode surface;negative: collection into a region of minimum field).

Localization and mixing of particles and cells can be achieved bycombining the above methods, as well as others. In the table below,examples of various localization/detector combinations are provided.Certain of the representative examples illustrate methods to localizeparticles or cells 2-dimensionally, allowing improvement in sensitivityor discrimination between different particles if an array of photondetectors (including a CCD) is used as opposed to a single photondetector (such as a PMT).

Method of Method of localizing Mixing: particles Example localizingcells particles or cells/means Detector centrifuge centrifuge centrifuge(long) cells/sediment single (short) (cent.) flow cell sediment andshallow channel particles/sediment single attach to surface above cells(grav.) flow cell (multiple sediment and shallow channelparticles/sediment imaging cell lines) attach to surface above cells(grav.) flow cell/magnetic sediment and localized particles (on imagingbead attach to surface magnetic bead beads)/sediment capture (grav.)flow cell/electric sediment and shallow channel particles/ single fieldattach to surface above cells electrophoresis tape/wick flow (into wick)air impact (tape) cells/sediment single (grav.) air impact centrifugeair impact (tape) cells/sediment single (short) (cent.) uniprep/magneticsediment to magnetic beads particles (on single bead surface on filtersurface beads)/sediment (grav.) flow past cells cells on filter flowpast cells single surface counter flow cells held on retained on filterparticles/flow past single filter surface by surface cells counter tocentrifugation cent. Force centrifuge tube centrifuge onto retained inflow cells/sediment single dielectrophoretic filter surface by (cent.)trap dielectrophorectic force traveling-wave sediment and traveling-waveparticles/sediment single dielectrophoresis attach to dielectrophoresis(grav.) traveling-wave dielectrophoresis dissolvable- separatecentrifuge (long) cells or single membrane tube compartment ontodissolvable particles/traveling- membrane wave dielectrophoresisacoustic/ultrasonic dissolve membrane and sediment (cent.)

Localization Examples

In each of the following examples, it is assumed, unless statedotherwise that the sample is an aliquot of aqueous solution compatiblewith short-term cell life and function, possibly containing targetparticles (though the descriptions below will assume the presence ofparticles). An aqueous sample can be obtained from environmental,clinical, air-to-liquid, washed-swab, or other samples. An air samplecan be obtained from a driven air stream (air sampler or surfacepickup), electrostatic capture, or settled airborne particles.References to cells should be understood to mean emitter cells in anaqueous media that is compatible with their life and function. Aparticle and cell brought into contact is assumed to result in emissionof one or more photons. A single or array photon detector existsexternal to the chamber in which the sample and cells are mixed, andthere may be additional optical elements to enhance capture anddetection of emitted photons (such as mirrors, lenses, lightpipes, etc.)either external or internal to the chamber. The chambers are eitherassumed to be transparent in part or in whole or to have another meansto allow emitted photons to reach the detector. Additional descriptionsof specific embodiments of the invention are provided in the Examples.

Centrifuge

A sample can be centrifuged in a chamber for a time sufficient tosediment the particles. Cells can be introduced to the chamber withoutdisturbing the particles and briefly centrifuged to sediment them ontothe particles. Photon detection can occur during or, more typically,after the spin.

Affinity Capture (Surface Capture)

A sample can be introduced into a microcentrifuge tube, multi-wellplate, filter unit, or other suitable device where some portion of thesurface in contact with the sample has been modified to be able to bindand retain particles that may be present in the sample through specificor non-specific binding interactions. Non-specific binding may befacilitated via electrostatic/ion-exchange interactions, hydrophobicinteractions, hydrophilic interactions, etc. Specific binding may befacilitated by immobilizing components to the surface that bind tosubstrates on the particles (e.g. antibodies, receptors, glycoproteins,proteins, peptides, carbohydrates, oligonucleotides, etc.), or byimmobilizing components that are bound by receptors on the surface ofparticles (small molecules, peptides, proteins, carbohydrates, etc.).

Affinity Capture (onto Mobile Substrate)

Similar to affinity capture on a surface, but particles are bound tomobile substrates (polymer beads, cells, charged molecules, magneticbeads, bacteria, etc.) that provide additional means of moving and/orlocalizing the particles or cells by various methods including thosedescribed herein.

Flow Cell

Emitter cells can be introduced to a shallow flow cell and allowed toattach to the bottom surface; non-adherent cells can be removed byadditional flow. A sample is introduced, displacing much of the cellmedia, and particles can sediment out onto the attached cells. Photonsare emitted as particles contact cells.

Flow Cell (Multiple Cell Lines)

Similar to the Flow Cell, with distinct regions of emitter cellsensitive to different target particles. Photon detection by imagingdetector to allow identification of which cells are stimulated, and,therefore, which target particles are present in the sample.

Flow Cell (Magnetic Bead)

This is similar to the Flow Cell. Appropriate magnetic beads are mixedwith the sample, allowing target particles to attach to the beads. Thesedecorated beads can be introduced to the flow cell where a stronglocalized magnetic field (due to a permanent magnet or electromagnet)captures them on the surface above the attached cells. Mixing can beinitiated by either removing the magnetic force and allow the beads tosediment onto the cells, or moving the magnetic force to attract thebeads to the surface to which the cells are attached.

Flow Cell (Electric Field)

Similar to Flow Cell, with the surface to which the cells attach and theone parallel to it being separate electrodes (at least one of whichmight be transparent). A sample can be introduced, displacing much ofthe cell media. An appropriate DC voltage is applied between theelectrodes and the particles are moved to the attached cells byelectrophoresis.

Tape/Wick

An air sample, possibly containing target particles, can be impacted ona transparent surface, which can be rigid or flexible (e.g., a tape),porous or nonporous. An absorbing material, or wick, can be attached,surrounding the impact area or, in the case of a porous surface, on theopposite side of that surface. Cells can be placed on the impact area,and, due to the wick, excess media will be absorbed, reducing the volumeand depth of the media bearing the cells and bringing them closer to theparticles. Cells sediment out onto the impacted particles or are,additionally, drawn toward them by flow if the surface is porous withthe wick material behind.

Air Impact

An air sample, possibly containing target particles, can be impactedinto a (fixed and initially empty) chamber which is suitable forcentrifugation. Cells can be introduced to the chamber withoutdisturbing the particles and briefly centrifuged to sediment them ontothe particles. Photon detection can occur without, during, or, moretypically, after the spin.

Filter Device/Magnetic Bead

A modified syringeless filter device, consisting of a chamber and aplunger with a suitable filter (Whatman™, Mini-Uniprep™, or similar),can be loaded with cells which are allowed to attach to the bottomsurface of the chamber; unattached cells can be washed away. A samplecan be introduced to the chamber along with magnetic beads with asuitable surface affinity. A modified plunger with a suitable magnetinserted inside and fixed near the back-side of the filter can beinserted into the chamber until the entrapped air escapes through thefilter. This assembly can be inverted and (possible after a time toallow the beads to sediment onto the filter's surface) the chamberpushed down onto the plunger. Magnetic beads and particles canaccumulate on the filter surface by filtration, sedimentation, andmagnetic attraction. Particles can attach to the magnetic beads or becaught among them. Upon re-inverting the assembly, the particles, areheld off the cells by the magnetic beads which, in turn, are held by themagnet inside the plunger. Removing that magnet releases the beads, andthe particles, which sediment across the short distance onto the cells.

Flow Past Cells

One or more layers of cells can be allowed to sediment onto the surfaceof a suitable filter or membrane at the bottom of a chamber. A samplecan be introduced to the chamber above the cells and pressure applied(by plunger or external pump, for example). As the sample flows past thecells, which are in intimate contact, particles are brought within closerange of the cells, allowing contact.

Counter Flow

One or more layers of cells can be allowed to sediment onto the surfaceof a suitable filter or membrane at the bottom of a ‘cell’ chamber. Asample can be placed in a separate ‘sample’ chamber which is connectedby some flow channel to the cell chamber at a point below the filter.The chambers can be arranged relative to one another such that, in acentrifuge, the sample chamber is closer to the axis of rotation; thelevel of the fluid in the sample chamber being closer to the axis ofrotation than the fluid in the cell chamber. By this means, during therotation of the centrifuge, fluid will flow between the chambers seekinga common distance from the axis of rotation. This can force some of thesample up through the filter supporting the cells and past the cellswhich are being held against that flow by the outward centrifugal force.As the sample flows past the cells, which are in intimate contact,particles are brought within close range of the cells, allowing contact.

Centrifuge Tube Filter

A sample can be introduced to the filter basket of a centrifuge tubefilter with a suitable size cutoff. Under appropriate centrifugeconditions, the sample will be forced through the filter, accumulatingparticles larger than the filter's cutoff size on the surface of thefilter. Cells can be added to the filter basket and be given a briefcentrifugation to bring them onto the filter surface and the particles.

Dielectrophoretic Trap

Similar to the Flow Cell, but with suitable electrodes on any of thesurfaces or projecting into the flow cell. A sample can be introduced bycontinuous flow past the electrodes, which can be connected to andelectrically driven by and external source. For a suitable combinationof flow rate, frequency, waveform, and amplitude, particles can beguided to and captured in a region of minimum electric field intensityabove the cells by negative dielectrophoresis. After stopping the flowand changing the electrical drive to the electrodes (possibly includinga DC voltage on between some electrodes to create an electrophoreticforce), the particle can sediment or be driven (by electrophoresis orpositive dielectrophoresis) onto the attached cells.

Traveling-Wave Dielectrophoresis

In a shallow cylindrical chamber, suitable electrodes (perhapstransparent) can be fabricated on one or both of the parallel faces,including a central planar electrode to collect particles, an electrodearound the periphery, and a set of spiral electrodes (either on the samesurface as the central one or the opposite surface). A sample can beintroduced to the chamber, and a DC potential applied between theperipheral and central electrodes to attract the particles to thecentral electrode by electrophoresis. By an exchange of fluids, cellscan be introduced to the chamber. Energizing the spiral electrodes withthe appropriate phase-shifted AC voltages can sweep the cells to thecenter by traveling-wave dielectrophoresis, where they can sediment ontothe particles.

Dissolvable-Membrane Tube

Use can be made of a electrically-actuated dissolvable gold membrane tomaintain isolation between target particles and emitter cells during thelocalization of the particles by centrifugal sedimentation. Either theparticles can be sedimented onto a membrane over the cells (as shown inFIG. 20), or the cells can be held off from the bottom of the chamber bya membrane spanning the bottom of a separate chamber (perhaps aninsert). In either case, after the membrane has be dissolved byelectrical activation, the particles and cells are mixed bysedimentation, possibly centrifugal.

Acoustics/Ultrasonics

Concentration of particles may be accomplished using acoustic orultrasonic signals. Particles can accumulate at nodes in a sanding wavepattern, or be move by a traveling-wave pattern. Cells can also be movedthis way, or delivered by any of several means discussed above.

Toxin Detection

In order to detect monovalent antigens, it is necessary to inducecrosslinking of surface antibodies using one of two general strategies.First, one can express two independent binding sites on the cellsurface, such that two receptor molecules can bind to a single ligand.Alternatively, one binding site can be expressed on the cell surface ifthe ligand is presented to the cell in a manner in which it appears tobe polyvalent. The following are specific examples using the model ofantibody-antigen recognition.

First, two antibodies can be expressed on the surface of a single cellline, each specific for different epitopes of a individual molecule(epitopes 1 and 2). The binding of a single molecule to two antibodies(one antibody against epitope 1 and another antibody against epitope 2)would initiate crosslinking and light emission. More specifically, asingle B cell line is engineered to express two independent antibodies,each recognizing a different epitope on a single molecule. The presenceof monomeric antigen is now capable of crosslinking the surfaceantibodies, resulting in increased intracellular Ca²⁺ and emission oflight by aequorin. A cell line that expresses functional antibodiesagainst both Y. pestis and F. tularensis (in addition to theendogenously expressed PC antibody) has been tested (see Examples). Eachof these agents is recognized independently by this cell line,indicating that both antibodies are functional and demonstrating thatemittor cells are capable of expressing two functional antibodiessimultaneously.

Another potential issue is the sensitivity of the optoelectronic deviceand methods with an antigen that cannot be pelleted using centrifugalforce. The Yersinia pestis F1 antigen exists as a low molecular weightpolymer in solution, and is therefore not sedimentable in our assay.However, B cells expressing antibody against F1 are capable of detectingsoluble F1 antigen at 5 ng/ml. This compares favorably with currentimmunoassay techniques and demonstrates that the optoelectronic devicecan be quite sensitive to soluble agents. A complementary experiment wascarried out using phosphorylcholine antigen conjugated to ovalbumin. Theability of this small antigen to stimulate antibody crosslinking on thecell surface indicates that this low molecular weight antigen,containing multiple copies of PC epitopes, is able to effectivelycrosslink surface antibodies and generate calcium influx and photonemission.

A second strategy can improve the limit of detection for monovalentantigens shown above by taking advantage of the centrifugal format. Thisapproach utilizes a scheme where one of the toxin antibodies isexpressed on the surface of benign bacteria and the second antibody onthe surface of B cells. The toxin can now be sedimented bycentrifugation, and B cells expressing the second antibody added.Because multiple antigens are immobilized on the surface of thebacteria, the toxin will in essence appear polyvalent to the B cell, andwill initiate a crosslinking event and photon emission. Morespecifically, Antibody against epitope 1 of a monomeric antigen (e.g.toxin) is expressed on the surface of bacteria. Soluble toxin binds tothese antibodies, coating the bacteria with toxin antigen. Thesetoxin-coated bacteria are sedimented by centrifugation prior to additionof B cells expressing antibody against epitope 2. Crosslinking of the Bcell antibodies results in light emission by aequorin. Experimentalresults on this strategy demonstrate the feasibility of detection ofbacterial surface antigens, and the increased sensitivity resulting fromsedimenting those bacteria prior to the addition of B cells. Similarapproaches can also be used for any poorly sedimenting agent to improveits presentation to B cells.

Crosslinking

Crosslinking of target particles can be achieved by any known means. Forexample, crosslinking can be achieved using one or more intermediateagents or molecules such as a peptide, an antibody, a chemical compound,an antibody, biotin, streptavidin, in addition, crosslinking can be viacovalent or non-covalent bonding. Methods for crosslinking also includeprecipitation or attachment to a solid phase via ligands, antibodies orchemical functional groups, as are known in the art.

Multiplexing Assays

The following is a description of how B cell mixtures can be used toincrease the number of detectable antigens without increasing the numberof detection channels (tubes, etc). The simplest way to detect multipleanalytes is to use a single emittor cell type per detection channel andto increase the number of cell assays by increasing the number ofdetection channels. This is acceptable for small numbers of assays but,as increasing numbers of analytes are added, the process becomes morecomplex and resource intensive. It is possible, however, to conduct upto 31 tests with concurrent negative controls in only a 5-channel systemif different B cell lines are mixed together.

As an example, if one has a single channel, one can at most detect asingle B cell assay. If, however, one has two channels, then one candetect 3 separate assays, where each channel contains an equal mixtureof 2 of the 3 separate B cell lines:

For example, if one has 3 B cell lines: A, B, C

And one mixes them into two channels thusly—

2 Channel 1: A, B Channel 2: B, C

Then there are three positive readout possibilities:

Channel 1 Channel 2 Yes No implies only A is present No Yes implies onlyC is present Yes Yes implies only B is present (or that more than oneagent is present, which we will consider unlikely for now)

Similarly, if one has 3 channels, one can detect 7 independent assays,by mixing groups of four cell lines together—

(A convenient shorthand will hereafter be utilized where the cell linesfor individual agents are labeled A through a letter corresponding tothe number of cell lines, and the channel numbers will be written toindicate what channels are required to detect positively for eachindividual agent as follows—123: F—means channels 1, 2, and 3 must allregister positive to ID agent F).

Channel 1 Channel 2 Channel 3 A, B, G, F B, C, E, F C, D, G, F 1: A 12:B 123: F 2: E 13: G 3: D 23: C

A formula embodying the relationship that simply describes the number ofindependent assays that can be accessed by a given number of channels,assuming all assays are mixed in equal proportion is:#Cell assays=2^(n)−1 where n is the number of channelsand the number of cell assays that need to be mixed in each channel isgiven by 2^((n-1)).

Thus, to mix 16 different B cell lines together, 5 channels are neededto interrogate 31 different assays. The design for a 10-channel systemcould, in fact, be used to provide ID for 31 separate agents withconcurrent negative controls (5-channel positive ID, 5-channel negativecontrol).

The channel mixtures and positive detection correlation for a 4-channelsystem (15 different assays) is shown below:

Channel 1 Channel 2 Channel 3 Channel 4 A, B, G, F, B, C, H, I F, C, D,I D, E, G, H I, K, L, M J, L, M, N J, K, M, O J, K, L, M 1: A 23: C 123:I 1234: M 2: N 24: H 234: J 3: O 34: D 134: K 4: E 12: B 124: L 13: F14: G

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the examples below, utilize thepresent invention to its fullest extent. The following examples are tobe construed as merely illustrative of how one skilled in the art canpractice the invention, and are not limitative of the remainder of thedisclosure in any way.

EXAMPLES

FIG. 1 is a schematic diagram showing the general cellular components ofthe invention. A cell (here a B cell) that contains an emitter molecule(here aequorin) has antibodies present on its surface. These antibodiesare specific for an antigen on a target particle, such as a biologicalwarfare agent. Binding of the target particle to antibodies on the Bcell brings two or more antibodies close together on the cell surface,causing a signal transduction cascade that leads to release of calciumfrom intracellular stores into the cytoplasm. This increase incytoplasmic calcium concentration causes aequorin to emit a photon. Thephoton is then captured and registered by a photo multiplier device,such as a CCD. Thus, a cellular biosensor can be implemented using cellshaving functional surface antibodies and containing a cytoplasmicemitter molecule that responds to increased calcium concentration.

Such a cell-based detection system provides rapid, sensitive, specific,accurate, and flexible detection of any antigen on any target particle.In regard to flexibility, the system can be modified to target anyparticle or groups of particles. In one example, a single emitter cellcan contain a plurality of antibody types, each type being specific fornon-overlapping groups of target particles. This single emitter cell canthen be used to identify a genus of target particle species at once.

In a second example, a reaction chamber can contain two types of emittercells. One type of emitter cell contains antibodies that are specificfor a genus of target particles (e.g., bacteria) and emits a photon of afirst wavelength in response to contact with any member of the genus.The second type of emitter cell contains antibodies that are specificfor a particular species within the genus (e.g., Yersinia pestis) andemits a photon of a second wavelength different from the firstwavelength in response to contact with the species. This arrangementoffers extremely high accuracy by reducing or eliminating false positivesignals. Only when photons of the first and second wavelength aredetected, would a positive event be registered. This nesting of emittercell specificities can be extended to more than two levels as necessaryto reduce or eliminate false positive signals.

FIG. 2 is a schematic diagram of a general architecture and useenvironment for the invention.

FIG. 3 is a schematic diagram of the molecular biology employed in oneembodiment of the invention. In this example, a universal B cell linethat expresses an emitter molecule (e.g., aequorin) but does not expressantibodies becomes the basis for generating B cells that can express anyantibody specific for any antigen. An antibody expression vector isintroduced into the universal B cell, selected for the presence of theexpression vector, and expanded for use in a detection system of theinvention. Using this strategy, in conjunction with pDisplay andVKExpress (described in “Antibodies” section above), target-specificemitter cells were generated for a variety of targets. Emitter cellsspecific for Foot and Mouth Disease virus (FMDV), Venezuelan EquineEncephalitis (VEE) virus, Yersinia pestis, Francisella tularensis,Brucella spp., the O1 and O139 strains of Vibrio cholera, and orthopoxviruses have been produced. The cDNA and sequence for the FMDV antibodyvariable regions were obtained from the USDA. The cDNAs and sequencesfor the Yersinia pestis, Francisella tularensis, Brucella spp., the 01and 0139 strains of Vibrio cholera antibody variable regions wereobtained from investigators at NMRC. The variable regions of the VEE andorthopox antibodies were cloned from hybridomas obtained from the CDCand USAMRIID, respectively. Foot and Mouth Disease Virus (FMDV),Yersinia pestis, Francisella tularensis, and Venezuelan EquineEncephalitis Virus (VEEV) are responsible for Foot and Mouth Disease,the Plague, tularemia, and encephalitis, respectively. Cloning from thehybridomas was done with a combination of primers described in severalpublished articles. Emitter cells specific for Bacillus globigii arebeing produced because this non-pathogenic bacterium is used by somemilitary agencies as a test organism in field trials of biologicalwarfare agent detection systems. FIG. 5 includes a line graph showingthe photon emission response when several clones of FMDV-specificemitter cells were contacted with live FMDV targets. In each case, theemitter cells fired photons within about 20-30 seconds after contactbetween the target and the cells. Included in the graph is data showinga lack of emission when a mutant FMDV (having single amino acid mutationin the viral coat protein) that would not be expected to bind to theemitter cell was contacted with an emitter cell clone. The negativecontrol supports the high specificity that is built into the detectionsystem.

Various configurations of a centrifuge and photomultiplier tube (PMT)arrangement can be incorporated into a system of the invention. Thearrangement includes a rotor (motor) that spins a sample microfuge tubefrom a swinging harness and includes a balance tube in a fixed position.The PMT is shown at the bottom, facing upwards toward the bottom end ofsample tube at rest. In a typical experiment for a target particle thatis smaller than the emitter cell, the particle-containing liquid sampleis placed in the sample tube and centrifuged under conditions sufficientto sediment the majority of the particles to the bottom of the tube(e.g., 60 seconds at 5600×g for Francisella tularensis). A suspension ofemitter cells is then layered onto the sample in the tube (so as not todisturb the sedimented particles) and spun briefly to pellet the cellsinto contact with the target particles. If target particles are presentin the candidate particles, photons of a specific wavelength should beemitted from the cells and captured and registered by the PMT.

In specific embodiments, the PMT can be a Hamamatsu HC 125-08 PMTinterfaced with a Stanford Research systems SR400 Two Channel GatedPhoton Counter. The centrifuge can be a Sapphire 17 turn, 18.5 AWG, 5amp motor having a swinging bucket configuration.

The centrifuge tube (reaction chamber) can be altered and upgraded asneeded to aid contact between candidate particles and the emitter cells.In one embodiment shown in FIG. 20, the tube contains an enclosedcompartment that holds pre-loaded emitter cells at the bottom of thetube. This compartment is separated from the rest of the tube by adissolvable gold anode membrane. In operation, a same containingcandidate particles is deposited into the tube and spun to concentratecandidate particles at the membrane. The membrane is then dissolved, andthe tube briefly spun to contact the particles with the emitter cells.This dissolvable membrane system is described by Langer and colleaguesin Angewantde Chimie International Edition 39:2396-2407, 2000; andNature 397:335-338, 1999.

The steps in the centrifuge process can be automated or alternativelydesigned so that the user need not stop the centrifuge at all. Forexample, the introduction and removal of liquids and samples can beaccomplished without the need to stop the rotor by adapting themechanical features of preparative centrifuges (e.g., ultracentrifuges)available from Beckman Instruments. In addition, it may be desirable todetect photon emission while centripetal forces are still being applied(e.g., when the contact between emitter cells and target particles areunstable without centrifugation). To detect photons emitted from thesample tube while it is spinning, the PMT can be arranged in a radialposition relative to the rotor axis. In most cases, the PMT in thisarrangement need not be spinning along with the sample tube, but insteadcan be stationary and simply register emission of photons when thesample tube passes in front of the PMT. If the emission signal is veryweak, then the detector (e.g., PMT, a CCD chip) can be coupled to therotor and spun along with the sample tube. Alternatively, multiple PMrscan be arrayed around a circumference of a rotor for detectingemissions.

If multiple samples are spun on the same rotor, the positioning orsignal processing of the PMT can be altered if necessary. In oneembodiment, the rotor accommodates 4 sample tubes, each containing cellsthat emit at the same wavelength. To differentiate emissions originatingfrom one sample over the emissions from another, a single radiallyaligned PMT can detect emissions continuously. The continuous emissiondata is then resolved using a timing trace from the rotor, whichmonitors the position of each sample over time, to allocate theemissions to each sample. Other variations are understood to be withinthe invention. For example, FIG. 17 is a schematic drawing of tworeaction tubes coupled to a rotor, with two PMTs aligned below thetubes. At a resting position, the rotor positions each of the tubesbelow the corresponding PMT, using the rotor position encoder. Inanother example, the centrifuge system shown in FIG. 17 can beintegrated with an air sample collector to achieve the system shown inFIG. 18. The radial aerosol impactor tube can include any type ofparticle monitor, such as described in U.S. Pat. No. 5,932,795 andreferences cited therein. In still another example, the system describedin FIG. 18 can be altered so that only one PMT aligned radially inrelation to the rotor axis is required, as shown in FIG. 19. Asdiscussed above, emissions registered by the PMT are resolved for eachsample tube using the shaft encoder.

Referring back to FIG. 17, fluid components including, but not limitedto, suspensions of B cells engineered to recognize specific bioagents,buffer solutions, preservatives, cell culture medium, can be placed ineach of several centrifuge tubes, mixed with a liquid suspension of thesample particles that has previously been collected from aerosol samplesin a separate process particles may include but are not limited to,proteins, peptides, chemicals, viruses, bacteria in vegetative and sporeforms, fungal spores, pollen grains, protozoa, blood or tissue derivedcells, and fragments thereof either alone or in conjunction with carrierparticles such as dust). When the spin motor is started, the centrifugetubes swing out into a radial position, and the B cells and/or sampleparticles are driven to the bottom of the centrifuge tubes at ratesdepending upon the size and density of the particles. The exact sequencewhereby cell and sample-containing fluids are added and centrifuged canbe customized based on their relative sedimentation velocities tomaximize signal. In general, it is expected that maximum photon outputcan be obtained from particles that sediment more slowly than B cells byspinning these samples (a pre-spin) for appropriate times before theaddition of B cells and spinning to bring them into contact. Forparticles sedimenting at similar or faster rates than B cells, a singlespin of the mixed sample and B cell components will initiate maximalphoton output from the system. Data from particle size analyzers(including but not limited to BAWS units, and fluid particle analyzers)incorporated upstream of the centrifugation device can be used toautomatically determine the optimal operation sequence and initiateappropriate computer-controlled automated sample handling.

When the “spin cycle” is terminated and the rotor comes to a controlledstop in a pre-determined position controlled by the spin motor and shaftencoder, the swing arms rotate under gravity forces so that the bottomsof the centrifuge tubes are presented to the sensitive surface of thephotomultiplier tubes, and any light signals are then recorded. In amodified version of this implementation, a single photomultiplier tubecan be positioned at the maximum radius of the rotor/tube configurationand used to collect photons from each tube as they pass by the sensitivesurface of the photomultiplier tube in succession. The photon outputmeasured from individual tubes can be assigned and combined based on themonitoring of the shaft encoding system.

Referring back to FIG. 18, the process of collection of the aerosolparticles is integrated with the process of bringing the aerosolparticles into contact with the B cells. Here, the centrifuge tubes areattached to swing arms that allow them to cover the ends of radialimpactor tubes while spinning, and the aerosol sample is induced to flowinto the sample inlet by the centrifugal forces acting on the air in therotating radial impactor tubes (can be assisted as necessary byadditional blower units). The high velocity of the flow causes aerosolparticles to impact on the inner surface of the centrifuge tube or thesurface of a liquid contained in the tubes and results in the capture ofthe particles on the surface of the tube or in the liquid, respectively.When a liquid is present, centrifugal pressures acting on the liquidwill balance the force imparted by the high velocity air flow requiredfor particle capture in the liquid and prevent it from being blown outby the impacting air. The aerosol particles are retained followingimpact with the tube surface or liquid and in the case of liquidcollection, forced to flow radially outward thereby providing increasedlocal particle concentrations at the maximum radius (the bottom of thecentrifuge tube). Addition of B cells and spinning them into the locallyconcentrated particle zone following the collection phase will initiatephoton output. Alternatively, the B cells can be present in the fluidduring collection and light output monitored in real time while spinningwith a single photomultiplier tube (FIG. 19). In a modified version ofthis implementation, the fluid components (including but not limited toparticle samples collected via an alternative bioaerosol collector, andsuspensions of engineered B cells) could be added to the inlet(s), andthe centrifugal forces can be used to distribute them to the tubes.

When the “spin cycle” is terminated and the rotor comes to a controlledstop in a pre-determined position controlled by the spin motor and shaftencoder, the swing arms rotate under gravity forces so that the bottomsof the centrifuge tubes are presented to the sensitive surface of thephoto multiplier tubes, and any light signals are then recorded. In amodified version of this implementation, a single photomultiplier tubecan be positioned at the maximum radius of the rotor/tube configurationand used to collect photons from each tube as they pass by the sensitivesurface of the photomultiplier tube in succession. The photon outputmeasured from individual tubes can be assigned and combined based on themonitoring of the shaft encoding system.

FIG. 7 is a schematic representation of the results of sequentialcentrifugations. For target particles smaller than emitter cells buthaving the same density of emitter cells, it is beneficial to first spinthe candidate particles (e.g., at high speed) to pellet them. Thereafterthe emitter cells can be added and spun under conditions which can bemilder to prevent reduction of their responsiveness as needed (topseries). In addition, this sequence of centrifugation forces almost allcandidate particles and emitter cells into a relatively small volume atthe bottom of a centrifuge tube. In contrast, mixing the candidateparticles and the emitter cells together and spinning them at one timewill lead to separation rather than contact between the particles andemitter cells (middle series). Of course, no spin at all dramaticallyreduces the effective concentration of particles and emitter cells inthe reaction (bottom series).

FIG. 8 includes a line graph showing in an actual experiment confirmingthe consequences proposed in FIG. 7. Emitter cells specific forFrancisella tularensis were mixed with killed Francisella tularensiscells in the three methods shown in FIG. 7. As seen in the line graph,the sequential spin method resulted in fast and efficient emission aftercontact. In contrast, the emission profile of the single spin method wasfar less pronounced in both timing and magnitude. The no-spin methodbarely exhibited a reaction.

A similar emission profile was generated in a separate experiment, assummarized in the line graph shown in FIG. 8. Inspection of the emissiontraces suggested that the single spin method resulted in target-specificemissions a little quicker than the two-spin method. However, thisresult was found to be primarily an artifact of the longer spin requiredfor the two-spin method and does not reflect an actual improvement inthe response time of the B cells. In fact, the initial slope of thetwo-spin method was significantly greater than that for the single spinmethod, indicating that the two-spin method led to a robust emitterresponse.

The sensitivity of the detection system shown in FIG. 8 was evaluated bytitrating the number of tularemia cells deposited into the centrifugetube. The results are summarized in the line graph shown in FIG. 10. Itappears that 25,000 emitter cells were capable of emitting photonsdetectable above background in response to about 5,300 tularemia targetparticles. It is expected that further optimization of reactionconditions will increase sensitivity.

Cell responses are improved after a single freeze-thaw cycle (see FIG.22). In this experiment, cells specific for Yersenia pestis (YP) werecentrifuged, resuspended in freezing medium (RPMI with 10% DMSO and anadditional 10% FBS), frozen at −80° C., and transferred to liquidnitrogen. Cells were thawed at 37° C. and 1 ml (2×10⁶) cells werediluted into to 4 mls of RPMI and incubated overnight at 37° C. Thefollowing day the cells were loaded with coelenterazine for 2 hours,washed into CO₂—Independent medium (CO₂—I) and recovered for 24 hours.10,000 cells were challenged with 5×10⁵ YP (50 ul of YP at 10⁷/ml).Untreated cells displayed a response of 9500 photons per second, whilefrozen thawed cells emitted approximately 6 fold more photons inresponse to YP. This stimulatory effect could be largely replicated byexposing the cells to freezing medium, without the actual freezing (5fold stimulation). It appears that the stimulatory factor in thefreezing medium is the DMSO. When cells were treated with 2% DMSO (thefinal concentration of DMSO when 1 ml of cells in freezing mediumcontaining 10% DMSO is diluted into 4 mls of normal medium) a similarlevel of stimulation was detected. The DMSO effect may be due to anumber of factors. DMSO is known to effect hematopoetic celldifferentiation, and may be stimulating the cells through this pathway.Additionally, cells frozen in medium containing glycerol also showsimilar levels of stimulation. Thus, it appears that the effect can alsoin part be due to a stress response induced by the DMSO and it can bepossible to replicate this stimulation using any of a number ofconditions that stimulate a “heat shock” response.

The cells can be stored frozen in the coelenterazine-charged state.Cells were loaded with coelenterazine, allowed to recover for 24 hours,and then frozen. Upon thawing the cells were washed through 10 ml ofCO₂—I medium and the cells were resuspended in CO₂I medium to aconcentration of 5×10⁵ cells/ml. These cells were capable of detectingYP (in this case about 1 hour after thawing, but shorter times arepossible). These cells remained capable of detecting agent for severaldays when stored at RT. Pretreatment of these cells with DMSO, prior toloading with coelenterazine and freezing, can increase the sensitivityof the cells to agent after thawing.

In FIG. 22, cells were challenged with 50 ul of 10,000,000 YP/ml dilutedin CO₂—I after various cell treatments. Untreated: Cells were grown inRPMI, loaded with coelenterazine, washed, recovered for 24 hours, andchallenged with YP. Freeze/Thaw: Cells were grown in RPMI, transferredto freezing medium, and frozen. Thawed cells (1 ml) were placed into 4mls of RPMI and incubated at 37° C. for 24 hours, loaded withcoelenterazine, washed, recovered for 24 hours, and challenged. FreezingMedium: Cells were grown in RPMI, transferred to freezing medium andincubated at RT for 10 minutes. Cells (1 ml) were placed into 4 mls ofRPMI and incubated at 37° C. for 24 hours, loaded with coelenterazine,washed, recovered for 24 hours, and challenged. 2% DMSO: Cells weregrown in RPMI, transferred to RPMI containing 2% DMSO and incubated at37E C for 24 hours, loaded with coelenterazine, washed, recovered for 24hours, and challenged.

A successful biological warfare detection system should be resistant tocontamination by common environmental substances present on abattlefield. To evaluate whether emitter cells can operate underenvironmental stress or contamination, emitter cells were mixed with atarget particle after exposure of the emitter cells to one hour of fullstrength diesel exhaust (left line graph in FIG. 11), or when theemitter cells were contaminated by 10⁷ E. coli (right line graph in FIG.11), which was used as a surrogate contaminant for any field bacterium.As shown in FIG. 11, the particular chemical and biological contaminantstested did not affect the ability of emitter cells to fire photons inresponse to a target particle.

FIGS. 13-14 describe a different embodiment of the invention that doesnot require centrifugation. The schematic diagram of FIG. 13 shows thevarious components of this embodiment. A biological aerosol warningsensor (BAWS) detects the present of particles, e.g., within apre-determined size range. An example of a BAWS is described inPrimmerman, Lincoln Laboratory Journal 12:3-32, 2000. If particlesmeeting specifications are detected, BAWS triggers an air-to-airconcentrator (specimen collector; as described in U.S. Pat. No.5,932,795) that allows particles of a particular size range to becollected and deposited in a well (reaction chamber) on a portion of aspecimen tape at a first station, which is shown in different views inFIG. 14. After candidate particles are deposited in the well, the tapeadvances to a second station under a reservoir of emitter cells and overa PMT. Emitter cells specific for a particular antigen on a targetparticles are deposited in the well, and the photon emission from thewell monitored.

During the time that candidate particles are detected by BAWS, thecandidate particles can be deposited on consecutive wells as the tape isadvanced through the first station (FIG. 14). In the second station, aplurality of emitter cell reservoirs, each containing emitter cellshaving different target specificities, are mounted on a turret thatrotates a particular reservoir into position to deposit differentemitter cells into the well. In this manner, different targets withinthe candidate particles can be detected, as shown in the schematic topview of the wells in FIG. 14. Of course, if the different emitter cellsemit at different wavelengths, it is possible to deposit the differentemitter cells into a single well containing candidate particles,provided that the PMT below the second station can distinguish photonsof different wavelengths.

FIG. 16 shows schematically yet another embodiment of a system of theinvention. In this embodiment, air particles are deposited in each ofsix wells within a row of a two-dimensional array (e.g., a tape having 6rows and hundreds of columns) at a first station. As the array isadvanced by one row, positioning the row in a second station, differentemitter cells are deposited into each well within the row, and emissionfrom all six reactions is detected simultaneously by a row PMTs underthe second station. To aid adhesion of particles to the wells on thetape, the wells can be coated with an adhesive or a liquid.

Particular Exemplifications

Methods and Materials

Cell Culture and Transfection

M12g3R cells were maintained at 37° C. in a humidified atmosphere of 5%CO₂ in RPMI 1640 supplemented with 10% fetal bovine serum, 1-mM sodiumpyruvate, 2-mM L-glutamine, 100-μM nonessential amino acids, 50-μM2-mercaptoethanol, 50-μg/ml streptomycin, and 50-U/mL penicillin (LifeTechnologies). Cells were transfected with linearized pCMV.AEQ.IRES.NEO[11] (20 μg of DNA per 10⁷ cells) via electroporation (270 V, 950 μF)and selected in 1-mg/mL G418 for 2 weeks. Antibiotic-resistant cellswere incubated in growth medium with 10 μM coelenterazine (MolecularProbes) for 2 h at room temperature, covered in foil, washed twice andresuspended in growth medium. The cells were screened for photonemission in response to anti-murine IgM F(ab′)₂ in a luminometer.

U937 cells were maintained in RPMI 1640 supplemented with 10% fetalbovine serum. The day before transfection cells were diluted to5×10⁶/mL. On the next day 2×10⁷ U937 cells were washed once in HBSS andresuspended in 900 μl of HBSS. Twenty micrograms of linearizedpCMV.AEQ.IRES.NEO was added to the cells and allowed to incubate for 10min at room temperature. The mixture was then transferred to anelectroporation cuvette (0.4 cm) and electroporated at 250 V and 975 μF.The cells were incubated in growth medium at 37° C. for 48 h, thencloned in medium containing 5-μg/ml Blasticidin by limiting dilution in96 well plates. After 10-14 days colonies were selected and grown up forscreening. Clones were loaded with coelenterazine and screened forresponse to 5-mM ionomycin. Positive clones were further expanded andcharacterized.

Antibody Expression Vectors

The light chain expression vector, VKExpress, contains the constantregion for the human kappa gene downstream of a multiple cloning site(MCS), under control of the human elongation factor-1α (EF-1α) promoter.

The heavy chain vector was generated by modifying pDisplay (Invitrogen),retaining the cytomegalovirus (CMV) promoter and leader sequence, butreplacing the platelet-derived growth factor receptor transmembranedomain with the gene for the membrane-bound constant region of murineIgM and removing both tags on either side of the MCS. The genomicsequence of the membrane-bound constant region of the murine IgM, CμM,was amplified by PCR using primers that contained EcoR I and Not I sites(5′ and 3′, respectively). The insert, prepared with a blunted EcoR Isite and digested with Not I, was cloned into pDisplay-hygro withblunted Bsm I and digested with Not I. The neomycin-resistance gene wasreplaced with one that confers resistance to hygromycin (Hygro®,obtained from pcDNA3.1 Hygro, Invitrogen) by adding Cla I and BstB Irestriction sites to the 5′ and 3′ ends of the Hygro® gene,respectively, by PCR, and cloning the new antibiotic-resistance geneinto those sites in pDisplay. The appropriate restriction sites areadded to the antibody variable regions using PCR, and the sequence ofall PCR products is confirmed before cloning into the expressionconstruct.

Cloning Antibody Genes

RNA was extracted with Trizol reagent (Life Technologies), according tothe manufacturer's recommendations, and first strand synthesis wasperformed using the Retroscript kit (Ambion). PCR was accomplished usingsets of primers designed to anneal to the leader sequences or theframework regions at the 5′ end, and the constant or framework regionsat the 3′ end. Cloning of the variable regions into the expressionvectors proceeded as follows. ApaL I and BamH I restriction sites wereadded to the 5′ and 3′ ends of the light chain variable regions by PCRwith primers containing those sequences, and cloned into VKExpress. Theheavy chain variable regions (V_(H)) were cloned into pDisplay-CμM in atwo-step process to eliminate the HA and myc tags. First, overlapextension PCR was used to fuse the V_(H) to the first 300 base pairs(bp) of CμM while, at the same time, adding a Bgl II restriction site tothe 5′ end. The insert was digested with Bgl II, which also cuts at by293 of the constant region, and cloned into pDis-CμM digested with thesame enzyme. A second overlap extension product fused the V_(H) to theIgK leader sequence, which was cloned in using the Kpn I and Bgl IIsites. We have subsequently modified this cloning process by producing apDisplay-CμM vector with a Bgl II site immediately following the leaderto allow for a single cloning step that eliminates both tags.

CANARY Assay

B cells were prepared for the luminescence assay by incubation in growthmedium with the addition of 2% DMSO at a concentration of 5×10⁵cells/mL. After 20-24 h, cells were incubated in the dark at roomtemperature for 2 h in assay medium [CO₂-Independent medium, 10% fetalbovine serum, 50-μg/ml streptomycin, 50-U/ml penicillin, and 250-ng/mLamphotericin B (Life Technologies)] with 50-μM coelenterazine (MolecularProbes, Eugene, Oreg.). The cells were then washed twice, resuspended inassay medium at a final concentration of 5×10⁵ cells/mL in 1.5-mLmicrocentrifuge tubes, and left to rotate overnight at room temperature.

Test samples were diluted in assay medium and centrifuged in 0.2-mL or1.5-mL tubes for 2 min in swing-bucket or horizontal centrifuge atmaximum speed. The B cells were gently mixed by inversion and 20 μl ofcells were deposited on the side of the sample tube. The sample tube wascentrifuged for 4 sec in a small, benchtop microfuge (Daigger) fittedwith a custom-made horizontal rotor, then inserted in the luminometer(Zylux, FB12). Responses were recorded using the Single Kinetic profileset for 1-sec intervals for a total of 60 sec. Positives were defined ashaving a signal-to-background ratio≧3 and a peak photon output withinthe range of 15-30 sec from the start of the 4-sec centrifugation.

U937 cells (5×10⁵ cells/ml) were incubated overnight with IFN gamma (200ng/mL, Sigma) at 37° C. The next day, 7.5×10⁵ cells were incubated for 2h in 100 μl of assay medium containing 200 μM of coelenterazine at roomtemperature in the dark, washed three times in assay medium, resuspendedat 5×10⁵ cells/mL, transferred to 1.5-ml tubes, and rotated overnight atroom temperature. Cells were incubated with antibody (10-100 μg/mL ofpurified, or a 1:1 ratio of hybridoma supernatant to cells) for 5-30 minat 37° C. then washed once and resuspended in assay medium. The assaywas performed as described above.

EGFP-Aequorin Expression Construct

To fuse aequorin to GFP we generated a construct containing the enhancedGFP (EGFP) gene fused to a 6 amino acid linker (SGGGSG), followed by theaequorin gene. EGFP was amplified by PCR from the pEGFP-C1 vector (BDBiosciences Clontech), removing the stop codon and adding the linkerregion to the 3′ end of the gene:

Sense primer: (SEQ ID NO: 12) 5′-GCCACCATGGTGAGCAAGGGC-3′Anti-sense primer: (SEQ ID NO: 15)5′-CCTGATCCACCGCCAGACTTGTACAGCTCGTCC-3′.

EGFP contains a double-amino-acid substitution (F64L and S65T) and showsenhanced fluorescence intensity compared to GFP. The aequorin gene wasamplified from pCMV Aequorin construct, adding the linker region to the5′ end of the gene: Sense primer:5′-CTGGCGGTGGATCAGGAATGACCAGCGAACAATA-3′ (SEQ ID NO: 22); Anti-senseprimer: 5′-TTAGGGGACAGCTCCA-3′ (SEQ ID NO 19). The EGFP and aequoringenes were then linked together by overlap extension PCR with the linkerregion serving as the overlap region. The fused genes were then clonedinto pcDNA3.1-TOPO (Invitrogen) and the sequence confirmed.

Assays for Clinical Samples

Nasal secretions were collected using foam-tipped swabs (VWR CriticalSwabs) then seeded with the indicated amount of B. anthracis spores andplaced in a basket containing a 5-μm filter (Millipore Ultrafree-MC)with 400 μL of assay medium. The eluate was collected in a 1.5-mLmicrofuge tube with a 2-min centrifugation, a step that also serves toconcentrate the spores to the bottom of the tube. After centrifugation,the basket and swab are removed and the assay performed in the sametube.

Human urine (3 mL), to which C. trachomatis EBs (10²-10⁵/mL, BiodesignInternational) had been added, was passed through a 5-μm syringe filter(Minisart). One half milliliter aliquots were centrifuged for 2 min in a1.5-mL microfuge tube at 10,000 RCF, the supernatant was decanted andthe residual allowed to wick away by placing the edge of the tubeagainst a clean paper towel. The pellet was resuspended by vortexing,0.5 mL of assay medium was added, and the sample centrifuged again for 2min at 10,000 RCF. The CANARY assay was performed as described above.

One-half milliliter of whole blood was collected into a custom-madeheparinized plasma separation tube and centrifuged for 90 sec at 3500RCF. The pathogen-containing plasma, with recovered volumes ranging from50 to 250 μL, was collected into an assay tube by inversion. Fiftymicroliters of the plasma was mixed with 0.5 mL of assay medium andtreated as described above in CANARY assay. To dilute the activatorpresent in human plasma, 450 μL was added to 50 μL of plasma. To removethe activator by adsorption, 50 μL of plasma was incubated with 50 μL(2×10⁵ cells) of the parental B-cell line, M12g3R, for 10 min at roomtemperature. The cells were sedimented with centrifugation at 1500 RCFfor 1 min to pellet cells, the plasma transferred to a clean tube andcentrifuged at maximum speed for 2 min.

To construct the device for intracellular pathogens in blood, 200 μL ofFICOLL™ HYPAQUE™ solution is placed in the bottom of a Capiject bloodcollection tube (T-M, Terumo Medical Corp.). The polyester gel from aCPT (Becton Dickinson Co.) is placed on top of the FICOLL. In order forproper separation of the blood cells to occur the whole blood must bediluted at least 6:1 with phosphate buffered saline (PBS); therefore 100μL of PBS is placed over the gel. Heparinized whole blood (600 μL) isplaced into the tube, the tube is inverted to mix the blood with the PBSand the device is centrifuged for 90 sec at 3500 RCF. The red plug inthe top of the device is replaced with an assay tube and the plasma andwhite blood cells are collected in the assay tube by inversion.Liberation of intracellular pathogens is achieved by adding 600 μL M-Percell-lysing reagent (Pierce Biotechnology, Inc.) to the assay tube andincubating at room temperature for 5 min with periodic vortexing. Thesample is centrifuged at 18,000 RCF for 1 min, the supernatant replacedwith 500 μL of assay medium, mixed by vortexing, and the centrifugationrepeated. The sample is analyzed for the presence of pathogen asdescribed above.

Chlamydia Validation

The following organisms were tested for cross reactivity with the C.trachomatis cell line: Pseudomonas aeruginosa, Streptococcus pyogenes,Enterococcus faecalis, Neisseria gonorrhoeae, Branhamella catarrhalis,Salmonella enteritidis, Escherichia coli, Klebsiella pneumoniae, Proteusmirabilis, Acinetobacter baumanii, Staphylococcus epidermidis,Streptococcus pneumoniae, Staphylococcus aureus, Candida albicans,Corynebacterium minutissimum, Lactobacillus acidophilus, Streptococcusagalactiae, Staphylococcus saprophyticus, Streptococcus group D,Streptococcus mutans, Garderella vaginalis, Gemella morbillorium.Serovars of C. trachomatis were obtained from Biodesign International.

Background

CANARY utilizes B cells that have been genetically engineered to produceaequorin, a calcium-sensitive bioluminescent protein originally found inthe Aequorea victoria jellyfish. The system works as follows: (1) Bcells can be exposed to suspected bioagents or other pathogens from anair sample, blood sample, or other source. (2) B cells have antibodiesspecific for certain bioagents. If one of those agents is present in thesample, it will bind to the antibodies on the surface of the B cell. (3)Crosslinking of a B cell's antibodies by a bioagent triggers anintracellular enzymatic cascade that releases calcium inside the cell.(4) In the presence of calcium, the aequorin emits blue-green light at469 nm. (5) Light from stimulated B cells can be detected using aphotomultiplier tube or other photodetector.

We have genetically engineered B-cell lines that express both (1)antibodies specific for bacterial and viral pathogens, and (2) theaequorin protein. Functional aequorin consists of the proteinapoaequorin and its substrate, coelenterazine, which is a chemical thatcan spontaneously cross the cell membrane and binds to apoaequorin.After binding calcium ions, aequorin undergoes a conformational changecausing the oxidation of coelenterazine and the emission of light.Activated aequorin-containing B cells, made antigen specific bytransfection with DNA expression vectors for recombinant antibodies,emit light when exposed to polyvalent antigen. When incorporated into anappropriate sensor format, these cells can be of tremendous benefit tomedical diagnostics, detection of biological warfare agents, andmonitoring of the quality of food, water, and air.

The B-cell detection system is intrinsically so fast (identification in<1 sec) that the primary delay in the assay is the time required tobring the pathogens in contact with the B cells. This problem is nottrivial, as the pathogens and B cells are essentially microscopicviscoelastic particles that tend to slide past each other in a fluidenvironment. We have solved this problem for bacteria and large virusesby using centrifugal force to drive the particles together. When theagent and B cells are simply placed together in suspension, the signalresponse is delayed in time and low in magnitude. When the agent and Bcells are pelleted by a 5-sec spin, both the speed and magnitude ofresponse improve. However, the greatest improvement in both speed andmagnitude occurs when the agent is pre-pelleted, prior to addition ofthe B cells. The B cells are then driven into the pellet by anadditional 5-sec spin.

Data was obtained for the bacterial pathogens Francisella tularensis andYersinia pestis using this centrifugal format. These data collectivelydemonstrate excellent specificity as well as the best combination ofspeed and sensitivity (50 cfu in approximately 3 min) of any knownpathogen identification method.

For larger viruses such as smallpox, which can be sedimented quickly atlow speeds, the present centrifugation method works well. However, wehave also engineered cell lines to produce antibodies specific forviruses such as FMD, Dengue, and VEE, which are too small to beconcentrated under the same conditions. Although the LOD for smallviruses is approximately 500,000 cfu in a 1-min assay, that number canbe improved by as much as 100 fold with longer centrifugation oraffinity purification.

CANARY B-Cell Sensor for Rapid, Sensitive Identification of Pathogens inClinical Samples

Introduction

We describe a novel sensor that provides the best combination of speedand sensitivity yet demonstrated for any pathogen-identificationtechnique. Our approach uses B lymphocytes, members of the immune systemthat have been optimized by nature to identify pathogens. We haveengineered B-cell lines to express cytosolic aequorin, acalcium-sensitive bioluminescent protein, as well as membrane-boundantibodies specific for pathogens of interest. The crosslinking ofmembrane-bound antibodies by a polyvalent antigen induces asignal-transduction cascade that sequentially involves tyrosine kinases,phospholipase C, and inositol triphosphate (IP₃). IP₃ activates calciumchannels, thereby increasing cytosolic calcium from both internal storesand the extracellular medium, which activates the aequorin, which emitslight. This sensor, which we call CANARY, can detect <50 cfu of pathogenin <3 min, including the time required to concentrate the samples. Incontrast, even state-of-the-art immunoassays take at least 15 min andhave a much higher limit of detection, and while the PCR can be bothhighly specific and sensitive, most reports cite protocols that take >30min. Although an ultrafast PCR with detection of 5 cfu in only 9 min hasbeen reported, even when coupled with the most rapid sample-preparationtechnology the total assay requires 20-30 min to complete. Because ofits unique combination of speed and sensitivity, CANARY couldrevolutionize pathogen identification in medical diagnostics, biowarfaredefense, food and water-quality monitoring, and other applications.

We first developed a genetic-engineering system that allows efficientproduction of a variety of B-cell lines. We generated a parental cellline with stable expression of cytosolic aequorin from the M12g3R (IgM+)B-cell line, selecting the clone with the maximum emission of light uponcrosslinking of the surface IgM. The M12g3R-aequorin cells aresubsequently transfected with plasmids containing antibody light andheavy chain constant regions, into which we insert the variable regionsspecific for a particular target. Clones from the second transfectionare selected based on their response to that target. In order to providean idea of the range of agents that can be identified using CANARY, wehave listed all of the 24 cell lines we have developed in the Tablebelow.

Detectable targets for CANARY cell lines Bacillus anthracis, Bacillussubtilis spores Potyvirus spores Salmonella spp. Phytophthora spp.Bacillus anthracis, Shigella dysenteriae Rift Valley Fever vegetative E.coli O157:H7 virus Francisella tularensis FMD virus Listeria spp.Yersinia pestis Dengue virus Listeria Vibrio cholerae O139Orthopoxviruses monocytogenes Vibrio cholerae O1 (smallpox) VEE virusBrucella spp. Ralstonia spp. Ovalbumin Chlamydia spp. Botulinum toxin

Results

The CANARY Assay

As little as 50 cfu of Yersinia pestis, the bacterium that causes theplague, is detected in less than 3-min total assay time. However, thereis no response to relatively large numbers of an unrelated pathogen,Francisella tularensis. Furthermore, even an overwhelming amount ofunrelated pathogen does not block the response to as few as 50 cfu of Y.pestis. In fact, for most bacteria or viruses large enough to beconcentrated in a microcentrifuge, we have observed similar levels ofsensitivity of ˜50 cfu or pfu. When the sensitivity of the Y.pestis-specific cell line was tested repeatedly over several months, theCANARY sensor could detect 20 cfu 62% of the time (n=73), 50 cfu 79% ofthe time (n=38), 200 (n=74) and 2000 cfu (n=71) 99% of the time, and20,000 cfu 100% of the time (n=66). A false-positive rate of only 0.4%(n=1288), combined with a level of sensitivity approaching that of PCRand an assay that can be performed in less than 3 min, makes CANARY oneof the most promising pathogen-identification technologies currently indevelopment.

Because the rate of diffusion determines the interactions between Bcells and non-sedimentable targets, the sensitivity of CANARY for smallviruses is higher than that for bacteria and large viruses. For example,B-cell lines specific for the A12 strain of foot-and-mouth disease virus(FMDV) produce an easily distinguishable signal when exposed to 7×10⁵plaque-forming units (pfu). Similarly, the sensitivity of the B-cellline specific for Venezuelan Equine Encephalitis (VEE) virus, testedwith strain TC-83 that had been titered prior to inactivation,demonstrates a detectable signal from 5×10⁵ pfu.

The specificity of CANARY is determined by the antibody the B cellsexpress, and can be as broad or narrow as the antibodies that areavailable. For example, while the FMDV cell line responds to wild typeA12 virus, no light is detected after the addition of an equivalentamount of the A12 variant strain (B2PD.3) that differs by three aminoacids, a change that disrupts the antibody-epitope interaction. Incontrast to the specificity of the FMDV cell lines, which react to onlyone strain of FMDV, the VEE B-cell line exhibits specificity similar tothat of the parent monoclonal antibody, reacting with VEE strainsrepresenting subtypes IA (TC-83, TRD), IB (PTF-39), IC (P676), ID(3880), and IE (Mena II). The M12g3R parental line (control B cells) wasalso tested for reactivity with the different strains of VEE, andalthough they exhibit a nonspecific signal in the presence of the TC-83and TRD antigen preparations (those isolated from suckling mouse brainbut not from tissue culture), the signal produced by the specific B-cellline is clearly distinguishable from that of the control (>10-fold). Wehave also produced a VEE B-cell line from hybridoma 1A4D-1, whichrecognizes all but the Mena II strain listed above. Therefore, givensuitable monoclonal antibodies, the specificity of the B-cell lines canbe designed to have either a broad or narrow range of reactivity basedon the antibodies that we choose to express. This greatly increases theflexibility of the system by designing it to distinguish organisms atthe genus, species, or subspecies level, depending on the application.

Improvement in Sensitivity for Small Viruses

Several methods of small virus concentration and sedimentation have beentested for their ability to improve CANARY responses to these agents.Precipitation with methanol, TCA, or sodium phosphotungstate did notimprove sensitivity, nor did absorption to nitrocellulose. Centrifugalconcentrators from various vendors appeared to bind nonspecifically tothe low concentrations of virus used in CANARY assays. Two methods havethus far shown good results: centrifugation and affinity purification.

Inactivated TC-83 VEE was used for all of the following virusconcentration experiments. To generate physiologically relevant virussamples, VEE aggregates were removed by passage through 0.1-μm syringefilters. Samples were then centrifuged for different times and analyzedby CANARY. Centrifugation for 1 min sedimented little virus, 5 min gaveintermediate results, and sedimentation approached completion in 10-30min. This pattern was in much closer agreement with theoreticalsedimentation rates for monomeric VEE, indicating that we have produceda test sample with similar sedimentation characteristics to thoseexpected for real samples. This also demonstrates that centrifugation ofmonomeric VEE for 10-30 min in a microfuge increases the signal, andtherefore the sensitivity.

Further experiments examined sonicating the aggregated material toimprove monomeric virus recovery. The LOD was improved in sonicatedsamples (500,000 pfu*) compared to the untreated sample (50,000,000pfu*), which reflects an increase in the quantity of monomeric viruspresent and able to pass through the 0.1-μm filter. The sample that wassonicated before filtering produced nearly 100 times more signal thanthe sample that was not sonicated. The sedimentation rate of themonomeric virus produced using sonication is also similar to thetheoretical sedimentation rates, indicating that sonication did notfragment the virus to an extent detectable in these assays.Centrifugation can improve the sensitivity by a factor of 100.

A second effective method for improving CANARY sensitivity to smallviruses is affinity purification. Monoclonal antibodies against VEE wereconjugated to protein G-coated magnetic beads. This affinity resin wasthen incubated with media containing VEE, the resin washed to removeunbound virus, and CANARY cells used to detect the virus attached to thesedimented resin. Incubation of VEE with these affinity resins for aslittle as 15 min clearly increased the amplitude of the CANARY cellresponse, and improved the LOD by a factor of 10.

Both affinity purification and centrifugation methods result in improvedCANARY responses to small virus. The method chosen will depend on thetype of sample to be examined. Samples containing sedimentable orsoluble interferents may be amenable to affinity purification of agentsusing magnetic beads. Samples containing soluble interferents or lackinginterferents altogether may be assayed using centrifugation protocols.

Rapid Cell Engineering

Generation of pathogen-specific CANARY cells requires an availablehybridoma cell line, involves several steps, and can take severalmonths. There is a need to develop a universal cell line that can beused to generate new pathogen-specific cells in a rapid (<1 day) butspecific manner, utilizing the CANARY platform. To address this issue,we explored using the Fc receptor as a possible “adaptor” molecule toattach pathogen-specific antibodies to CANARY cells. The Fc receptorsare a family of membrane-expressed proteins that bind to antibodies orimmune complexes. They are expressed on several hematopoietic cellsincluding monocytes and macrophages. Several subclasses of Fc receptorsexist including Fcγ Receptor I (FcγRI), a high-affinity binder ofsoluble antibody. FcγRI binds to the constant region (Fc portion) ofImmunoglobulin G (IgG) leaving the antigen-binding region of theantibody free. Crosslinking of the antibody-bound receptor by specificantigen initiates a signaling pathway that stimulates calcium release.

The human macrophage cell line, U937, contains endogenous FCγR1 whichcan be upregulated upon treatment with IFNγ. Initial experimentsdemonstrate that U937 cells can be engineered to rapidly to respond toseveral different pathogens or simulants. U937 cells were treated for 24h with IFNγ (200 ng/ml) to increase expression of endogenous FcγRI, andprepared for the CANARY assay. Then the cells were incubated with thefollowing antibodies: mouse anti-B. anthracis spore, rabbit polyclonalanti-B. anthracis spore, mouse anti-F. tularensis, or mouse anti-B.subtilis. Cells were then used in the standard CANARY assay where theydetected as few as 1000 cfu B. anthracis spores with the monoclonalantibody and 10,000 cfu spores with the rabbit polyclonal, as well as10,000 cfu F. tularensis and 1,000 cfu B. subtilis spores. Although notas sensitive as the genetically engineered B cells, we have demonstratedthe development of a rapidly engineered CANARY cell that requires daysinstead of months.

Multiplexed Assays

We have evaluated the feasibility of combining several different B-celllines in a single assay. This would allow the detection of severalagents with a single test, though it would not distinguish which agentis in the sample. Detection of 3 different agents with a single cellreagent was demonstrated where the limit of detection for B. anthraciswas 50 cfu of B.a., Y. pestis was 50 cfu of Y.p., and F. tularensis was500 cfu of F.t. At an optimized cell concentration and amount of 40 μLof 1.25×10⁵ cells/mL, we were able to show that 4 cell lines can becombined without any loss in sensitivity.

A second method of multiplexing is cell lines that express more than oneantibody and can respond to more than one agent. We have generated acell line that expresses two antibodies, one specific for B. anthracisspores and the other for Y. pestis. This cell line was used to detectonly 50 cfu of either B. anthracis spores or Y. pestis, demonstratingthat we can create a cell line with multiple detection capabilitieswithout any loss in sensitivity.

A third method that provides a multiplexed assay is CANARY cell linesthat emit light of different wavelengths. In the jellyfish Aequoreavictoria aequorin is naturally associated with green fluorescent protein(GFP). When the aequorin binds calcium and oxidizes coelenterazine ittransfers its energy to GFP and stimulates the emission of green light(λmax, 509 nm). This naturally occurring chemiluminescence resonanceenergy transfer (CRET) activity can be reproduced in vitro by fusing theaequorin protein to GFP. GFP can be genetically modified to producevarious fluorescent proteins including cyan fluorescent protein andyellow fluorescent protein. Fusion of aequorin to different GFPconstructs can generate several aequorin proteins capable of producinglight of different wavelengths. CANARY cells expressing theseaequorin-GFP proteins provide a multiplexed assay, where detection ofone or more wavelengths allows the identification of several pathogensin a single assay. This type of multiplexed assay has severaladvantages, including the identification of several pathogens in asingle assay when the sample size is limited, the ability to test formultiple pathogens at one time when using a single channel sensor, andthe potential to decrease false-positive rates in multi-channel sensorsby increasing the number of replicates.

The EGFP-aequorin construct was transfected into M12g3R murine B cellsand the clones were screened by response to anti-IgM stimulation.Positive clones were analyzed on a flow cytometer where cells expressingEGFP (λmax, 509 nm) can be detected in the FL1 channel, which measureslight in the green spectrum from 515 to 545 nm. In order to furtherdemonstrate that the cells expressing EGFP-aequorin are emitting adifferent wavelength of light than those expressing wild-type aequorin,we analyzed the light output with two photomultiplier tubes (PMTs) withdifferent band-pass filters, 480 nm and 510 nm. The cells werestimulated with anti-IgM, and the light was measured simultaneously byboth PMTs. Because the emission spectra of aequorin and theEGFP-aequorin overlap, the results are expressed as the ratio ofgreen/blue light. The amount of green light emitted by the cellsexpressing EGFP-aequorin was significantly higher than that emitted bythe cells expressing wild-type aequorin. Interestingly, unlike wild-typeEGFP that fluoresces in the absence of any cofactor, the EGFP-aequorinrequired the presence of the aequorin cofactor, coelenterazine, beforefluorescence was observed.

Development of Assays for Clinical Samples

There are many applications where a rapid pathogen-identificationtechnique would be extremely valuable. For instance, a rapid test wouldensure timely, accurate treatment of patients in the early stages ofinfection where immediate treatment is important, as in the case ofinhalation anthrax. We therefore investigated the use of CANARY fordetecting pathogens in clinically relevant samples. As few as 50 cfu ofB. anthracis spores added to nasal swabs prior to sample preparation canbe detected. In this protocol the swabs were placed in a basketcontaining a 5-μm filter with 400 μL of assay medium. The eluate wascollected in a 1.5-mL microfuge tube with a 2-min centrifugation, a stepthat also serves to concentrate the spores to the bottom of the tube.After centrifugation, the basket and swab are removed and the assayperformed in the same tube. The total assay time is less than 5 min, andthus, CANARY provides an excellent first screen for people who may havebeen exposed to aerosolized B. anthracis spores, thereby allowingimmediate treatment.

Another example is the need for rapid point-of-care diagnostic tests toensure treatment and control of diseases, such as those that aresexually transmitted, for which there is a high rate of medicationnoncompliance. C. trachomatis is a sexually transmitted disease that hasa high prevalence, can cause pelvic inflammatory disease and fertilityproblems, and is underdiagnosed because of the high number ofasymptomatic cases. Historically, the disease has been diagnosed fromcervical or urethral smears with tests that require considerable timeand expertise. Although the elementary bodies (EBs) of the organism canbe found in the urine, a less invasive sample to collect, it is presentin such low numbers that, until recently, the only tests sensitiveenough to be effective are those that amplify nucleic acids. In a recentreport, the concentration of C. trachomatis in urine from infectedpatients was determined to range from 30 to 2×10⁵ EBs/mL using aquantitative ligase chain reaction, an assay that takes several hours toperform (Abbott). Due to the rapid performance of CANARY, we were ableto demonstrate detection of 500 C. trachomatis EBs in urine in less than5 min. Thus, CANARY is also useful as a rapid, sensitive assay for thediagnosis of C. trachomatis infections in a noninvasive test.

Whole blood is a difficult matrix to assay because of its opacity andthe presence of both activators and inhibitors of the CANARY assay. Themethod we have developed relies on the use of plasma-separation tubes(PST) and differential centrifugation. This process uses a thixotropicgel with a density between that of plasma and blood cells, which forms abarrier between the plasma and cells during centrifugation. Any bacteriaor viruses present in the blood remain in the plasma phase aftercentrifugation, which can then be harvested and tested in CANARY. Usinga device assembled from commercial off-the-shelf (“COTS”) parts, we havedemonstrated the separation of whole blood samples in three rapid,simple steps. One-half milliliter of whole blood is collected into aheparinized plasma separation tube (step 1) and centrifuged for 90 sec(step 2). The separated pathogen-containing plasma, with recoveredvolume ranging from 50 to 250 μL, is collected into an assay tube byinversion (step 3). 50 μL of the plasma is mixed with 0.5 mL of assaymedium (a process that reduces the effect of a CANARY cell activatorthat is present in plasma, as explained in more detail below) and themixture is centrifuged to pellet the pathogen. The sample is then testedwith pathogen-specific CANARY cells. The total time required from bloodcollection to pathogen detection is ˜5 min. Using the PST method, theLOD is ˜1000 cfu of live, avirulent Y. pestis/mL whole blood. By using50 μL of the 200 μL of plasma recovered from 0.5 mL of whole blood, wedetected as little as 125 cfu (assuming full recovery) per whole-bloodsample. These results were consistent for each donor tested to date.

As mentioned previously, human plasma contains a B-cell activator thatinterferes with the CANARY assay, making it difficult to get a clearsignal from low concentrations of agent that can be differentiated fromthe background. The signal produced by the activator peaks later than apathogen-induced signal, and the amplitude of the signal is donordependent, ranging from barely perceptible to several orders ofmagnitude. We have developed three sample-preparation methods thateffectively remove the activator. Method 1 takes advantage of the factthat the activator is soluble and can therefore be removed by replacingthe plasma with assay buffer. This technique is effective with bacteriaand large viruses that can be sedimented by centrifugation beforereplacement, but is not useful with small viruses or soluble proteins.Method 2 involves diluting out the effect of the activator by adding anexcess of CANARY assay medium to the plasma sample. This method is themost rapid and simple but needs further testing to ensure itseffectiveness with a variety of blood samples, particularly those whichcontain a high-level activator. Method 3 utilizes a pretreatment of theplasma sample with B cells that function as an adsorbent for theactivator.

In order to detect intracellular pathogens in white blood cells, amethod was developed which incorporated modifications to the prototypedevice designed to detect pathogens in plasma. These modifications arebased on a commercially available blood vacutainer tube, CellPreparation Tube (CPT). This tube was designed to collect whole bloodand separate mononuclear white blood cells by combining a polyester geland a density-gradient cell-isolation medium in a single tube. Cellseparation occurs during a single centrifugation step. The disadvantagesof the commercial tube are that they require at least 6 mL of blood anda minimum of 15-min centrifugation. By incorporating the CPT gel anddensity-gradient medium into the custom-made processing device describedabove, the amount of blood is reduced to 0.5 mL and the centrifugationtime is only 90 sec.

In order for proper separation of the blood cells to occur the wholeblood should be diluted at least 6:1 with phosphate buffered saline(PBS); therefore 100 μL of PBS is placed over the gel. The device is nowready to process a blood sample. Heparinized whole blood (600 μL) isplaced into the tube, the tube is inverted to mix the blood with thePBS. After a 90-sec centrifugation, the blood separates into its variouscomponents. The red plug in the top of the device is replaced with anassay tube and the plasma and white blood cells are collected in theassay tube by inversion.

Liberation of intracellular pathogens is achieved by adding M-Percell-lysing reagent to the assay tube and incubating at room temperaturefor 5 min with periodic vortexing. The sample is centrifuged for 1 minto sediment the pathogen, the supernatant is replaced with 500 μL ofassay medium, mixed by vortexing, and the centrifugation repeated. Thetotal time from blood collection to agent detection is ˜12 min.Detection of 1000 cfu of live Y. pestis per mL of whole blood (600cfu/assay) was achieved. This method should work well for detection ofintracellular pathogens that can be concentrated by low-speedcentrifugation (i.e., bacteria and large viruses).

Validation

Validation was performed in which both cross reactivity and sensitivityusing the C. trachomatis cell line was tested. Cross reactivity wasobserved with only 2 of the 22 types of bacteria tested, and only atvery high concentrations (10⁷/mL). While Streptococcus pneumoniaebacteria produce a positive reaction, it is only the monomericpolysaccharide of the bacteria that appears in the urine of a patientwith pneumonia, and monomeric antigens do not stimulate the CANARY Bcells. The other bacteria that cross reacted, Gemella morbillorum, is anormal intestinal organism that may contaminate a urine sample, but isunlikely to at such a high concentration. The sensitivity of the C.trachomatis cell line ranged from 10 to 150 EBs (10, 50, and 150 forserovars D, H, and K, respectively), depending on the serovar of C.trachomatis tested. However, since different lots gave slightlydifferent results, the range of sensitivity may have been due to theaccuracy of the quantitation and not differential response of the cellline. In either case, the LOD determined by the validation was in therange of 10's to 100's.

Conclusion

The CANARY B-cell-based biosensor exploits a highly evolved system forpathogen identification that provides several advantages over otheridentification technologies. With CANARY it is possible to provideidentification in less than 5 min, and with those pathogens large enoughto be concentrated in a microfuge, we have demonstrated a level ofsensitivity that approaches PCR. In comparison, state-of-the-artimmunoassays require at least 14 min and have a higher limit ofdetection (6×10⁴ cfu or 6×10⁶ pfu). While PCR is extremely sensitive (1to 5 cfu), highly specific, and has enjoyed technological breakthroughsthat have reduced the time for amplification and signal detection, theassay takes at least 7 min (but typically 20-30 min), not including thetime required to extract and purify the DNA. Applications that wouldbenefit from such a technology include point-of-care diagnostics forillnesses where the return rate for treatment is low but the societalimpact is high, such as sexually transmitted diseases. In addition,CANARY would be valuable for detection of agricultural pathogens atports of entry, pre-symptomatic detection from nasal swabs in theaftermath of a biowarfare attack, or screening of perishable foodsupplies. In fact, CANARY is a rapid, sensitive method that can enablethe detection and identification of highly infectious pathogens in anytime-critical setting.

Dielectrophoresis for Concentration of Small Particles

Introduction: The CANARY assay can use centrifugation as a key step incolocalizing antigen-containing particles prior to introducing them tothe B cells for recognition and signal generation. This approach hasbeen highly successful in the rapid detection of bacterial and viraltargets which have particle sizes of greater than 500 nm; however someviral targets, being much smaller, are more difficult to concentrate inthis manner and can require more extensive centrifugation at very highspeeds, and/or the addition of steps such as intermediate binding of thetarget particles to beads, to improve sedimentation of the compositeparticles. In order to determine centrifugation velocities required tosediment particles of a given size, we can use Stokes law ofsedimentation (Equation 1) to calculate a particle's velocity in a fluidas a function of fluid viscosity and rotational parameters.

$v_{p} = {\frac{2}{9}{r_{p}^{2}\left( \frac{\rho_{p} - \rho_{m}}{\mu} \right)}{D\left( \frac{2\pi\; N}{60} \right)}^{2}}$

-   -   v_(p)=particle velocity    -   r_(p)=particle radius    -   ρ=density    -   μ=fluid viscosity    -   D=centrifugal radius    -   N=rotational speed

As an example, using the current benchtop CANARY centrifuge having amaximum speed of 18,800 rpm, concentration of VEE viral particles(diameter 70 nm) through a typical sample volume of 50 μL in amicrocentrifuge tube would take approximately 15 min. Using anultracentrifuge with spin speeds of up to 100,000 rpm could reduce thissedimentation time to less than 1 min, but with the associatedcomplexity in required equipment.

We have developed non-centrifuge-based methods for small-particleconcentration, one being electrokinetic or electric-field-based methods.The most well-known of these techniques is electrophoresis, which hasbeen used very successfully for many years to manipulate and separatecharged particles and large molecules, including DNA and proteins, inliquids and gel-based media. It relies on the application of an electricfield across the medium in which the particles reside; under theinfluence of this (constant) field, charged particles will migrate toone of the electrodes. The direction and rate of migration of theparticles depends on their charge and size, as well as the properties ofthe medium, including its pH and ionic strength. Electrophoresis is ahighly useful technique for the manipulation charged particles in arelatively imprecise manner. However, to concentrate particles toparticular locations, and additionally these particles are notnecessarily charged but are polarizable, then a technique calleddielectrophoresis is used.

Dielectrophoresis—The Basics

The term dielectrophoresis (DEP) was first used by Pohl, who was able toinduce movement and separation of multiple cell types by using anonuniform electric field to generate a charge separation (polarization,creating a dipole) in uncharged particles. There are two DEP modes,positive and negative, as illustrated in FIG. 181; the mode isdetermined by the relative polarizability of the particle with respectto the surrounding medium. In both cases, the particle and the mediumundergo a charge separation under an applied nonuniform electric field.If the particle is more polarizable than the medium, the net dipoleresults in the attraction of the particle towards the region of highestelectric field; this is referred to as positive DEP. If the particle isless polarizable than the medium, then the fluid medium migrates towardsthe high field region, which pushes the particle towards theelectric-field minimum; this is negative DEP.

Note that if the polarity of the electric field is switched, the inducedcharges and dipoles also switch polarities, so that the particle stillmoves in the same direction; this enables the use of alternating current(AC) fields to manipulate the particle. AC fields allow the exploitationof the polarizability of a particle which is frequency dependent; thismeans that the same particle can undergo either positive or negativeDEP, depending on the frequency of the applied field. AC fields are alsomore desirable than DC because they do not result in significant net gasgeneration at the electrodes due to electrolysis. Generally, at lowfrequencies a particle will experience positive dielectrophoresis, sincethere is enough time in each cycle for the charges in the particle toseparate with respect to the charges in the medium. At higherfrequencies, charge distribution inside the particle cannot “keep up”,and the particle becomes less polarizable with respect to the medium,putting it into the negative dielectrophoresis regime. Positive DEP canbe used to concentrate particles at electrodes, and negative DEP to trapthem in electric field “wells” away from electrodes. The frequency atwhich a particle switches from positive to negative DEP is called thecrossover frequency.

Equation 2 shows the factors that influence the dielectrophoretic force(F_(DEP)); the force is proportional to the square of the appliedvoltage (V) and the cube of the particle radius (r), and inverselyproportional to the electrode spacing (d). It is also a function of therelative permittivities of the particle (ε_(p)*) and the medium(ε_(m)*), both of which are frequency (ω) dependent, as indicated bytheir effect on the Clausius-Mossotti factor K(ω).

F_(DEP) = 2 π r³ɛ_(m)Re[K(ω)]∇E²${\nabla E^{2}}\alpha\frac{V^{2}}{d^{3}}$${K(\omega)} = \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}}$

There are many demonstrations of the use of positive and negative DEP tomanipulate and trap cells and larger (>1 μm) particles. More recently,with advances in fabrication methods that enable the formation ofsmaller-geometry electrodes, DEP has also been used to trap largeviruses and even macromolecules such as proteins and DNA. We are usingDEP to concentrate particles of less than 100 nm in diameter, achallenging problem as Equation 2 clearly indicates that as particlesize decreases, there is a need to substantially increase the appliedelectric field (possibly generating electrolysis) and/or decrease theelectrode spacing and geometry (which complicates fabricationprocesses).

Materials and Methods

Design of DEP Chip

A set of devices with various geometries of interdigitated electrodeswere fabricated. Each device consisted of a set of platinum linesdeposited on a square quartz chip with dimensions 25 mm×25 mm×0.5 mm,with the electrode pattern defined using a conventional liftoff processin which the negative image of the metal pattern is formedphotolithographically using a photosensitive polymer, after whichplatinum is deposited using electron-beam evaporation and the excessplatinum is removed by dissolving the underlying photopolymer (FIG.182). FIG. 183 shows the basic electrode pattern and a completed devicechip. In addition to the linear two-electrode pattern, a small number ofspiral four-electrode structures were also fabricated; these may be usedto achieve traveling-wave dielectrophoresis, in which four AC signals,separated equally in phase, are applied to the electrodes to step theparticles along the structure to the center of the spiral.

Test Setup

Two setups were used to exercise the DEP chips, one in which the chipswere held horizontally and the other in which they were held vertically.Note that although the simulations used a two-chip structure, initialexperiments used a single set of electrodes only, to demonstrateattraction and repulsion of the test particles via positive and negativeDEP respectively. In both the horizontal and the vertical configuration,the fluid channel was formed by sandwiching a 125-μm-thick siliconegasket between the electrode-containing chip and a plain quartz chip.The device was held in one of two types of jigs, and electrical accesswas obtained via copper alligator clips that contacted metal padsconnected to the interdigitated electrodes on the electrode-containingchip.

Bead movement was generated by applying a square wave across the twoelectrodes, of amplitude 1-10 V (peak to peak) at a frequency of 1 Hz to10 MHz, using a Hewlett Packard HP237 function generator. Test particlesconsisted of fluorescently tagged polystyrene beads (Bangs Laboratories,emission at 655 nm) of various diameters, suspended in distilled water.In the horizontal configuration, bead motion was observed in a staticmode by filling the channel with beads suspended in fluid, applying thefield, and imaging bead movement. In the vertical configuration, fluidflow was generated by applying a small amount of absorbent material atone end of the channel to act as a wick. Images of the particles werecaptured using a CCD camera attached to an Olympus BX60 fluorescencemicroscope equipped with a variety of fluorescence filter sets, andrecorded on a DVD recorder.

Results

The goal of this effort was to show the ability to localize smallparticles using DEP. Therefore the devices were evaluated for thisability in either positive or negative DEP mode. At low frequencies thebeads exhibited positive DEP, in which the beads localized to theelectrodes; as the excitation frequency was increased, at some point thebeads were released from the electrode surface and started to move awayfrom the electrodes.

Using the horizontal configuration we were able to show attraction andrepulsion of 2.7-μm and 0.3-μm-diameter beads using electrodes with 5-μmlinewidths, but were unable to determine the repulsion distance due tothe configuration of the test setup, in which the chips were heldhorizontally and observed from above. Subsequently fabricated devicescontaining electrodes with 2-μm linewidths were made and tested in thevertical configuration.

Conclusions

We were able to demonstrate both positive and negative dielectrophoreticmovement of 300-nm and larger particles using interdigitated metalelectrodes with linewidths of 2 μm. In the negative DEP regime,particles were repelled from the electrode plane to a distance of up to20 μm. Negative DEP was also demonstrated using 50-nm particles, butwith a repulsion distance of only 5 μm. The eventual goal of this effortwas to concentrate particles smaller than 100 nm in diameter, andfurthermore, to be able to repel them a suitable distance away from thedriving electrodes to be able to separate the concentrated plane ofparticles from the remainder of the sample fluid. A repulsion distanceof at least 100 μm would facilitate this separation in a microfluidicchannel, but in our devices we were able to achieve a repulsion distanceof less than 20 μm. If we look at the parameters governing the effectiveDEP force, we find that it scales as the inverse cube of the electrodelinewidth. This indicates that a 10× reduction in the linewidth shouldgive a 1000× increase in DEP force, and a corresponding increase inrepulsion distance for a given driving voltage and particle diameter.Electrodes with 0.2-μm linewidths can be fabricated using the advancedphotolithography systems, and these devices will enable concentration of50-nm particles.

Toxin Detection with CANARY

Methods and Materials

GST-BoNT/A and E Hc Recombinant Expression and Purification

cDNAs encoding BoNT/A Hc and BoNT/E Hc in plasmid pGEX-4T3. Plasmidswere transfected into BL21 (DE3) pLys (Invitrogen) according to themanufacturer's instructions. Bacteria harboring plasmid were dilutedfrom overnight cultures and grown to an OD600 of ˜0.5, IPTG was added toa final concentration of 400 μM, and the incubation continued at 30° C.for 4 h. Bacteria were harvested, and each liter resuspended in 30 mL ofBugBuster with 30 μL of benzonase nuclease (Novagen), and the tuberotated at RT for 20 min. The lysate was centrifuged at 21,000 RCF for30 min at 4° C., the soluble protein decanted onto 3 mL of glutathionesepharose (Amersham Biosciences) equilibrated with PBS, 1 mM EDTA. Theslurry was rotated at 4° C. for 2 h and poured onto a 20 mL disposablecolumn (BioRad). The column was washed with PBS/EDTA, and recombinantprotein eluted in 10-mM glutathione in 100-mM Tris, pH 8.0.

Nonmedical Matrices

1/7th volume of 7× HNa (560-mM NaCl, 1.4-M Hepes pH 7.9, andantibody-coated beads) was added to antigen-spiked solution. At the endof a 12-min binding step, 190 μL of assay medium was added, the tube wasplaced on the magnet for 30 sec, and the supernatant discarded. Thebeads were resuspended in 50 μL of assay medium, 20 μL of cells wereadded, the tube was spun for 5 sec to sediment the beads and CANARYcells, and light output monitored on a luminometer.

Antibody Production

Hybridomas were acclimatized to Hybridoma SFM media (Gibco)+1×nonessential amino acids (Gibco, 100-μM Na Pyruvate, and 200-μML-glutamine). Some hybridomas required 10% serum initially, but allantibodies were ultimately produced in 0% serum containing media.

Antibody Purification

Hybridoma supernatants produced in serum-free media were centrifuged at3700 RPM in clinical centrifuge, and the supernatant 0.2-micronfiltered. 1 mL of PBS-equilibrated Protein G Sepharose 4 Fast Flow (GEHealthcare) was added to supernatant and rotated slowly either 3-4 h atRT or overnight at 4° C. Resin was poured into disposable column, washedwith PBS, and 1 mL fractions eluted with 100 mM KPO4 pH 2.7 directlyinto 100 μL of 1 M Hepes pH 8.5. Buffer was exchanged to PBS using NAP-5columns.

Crosslinking to Protein G Resin

Beads (Dynal Dynabeads Protein G) were washed into 50 mM NaOAc, pH 5.0.The pH of the hybridoma supernatant was brought to about 5.0, BSA addedto 0.1%, and to solution filtered through a 0.2-micron filter. 100 μL ofbeads were added to 10 mL of hybridoma supernatant, and the tube rotatedfor 1 hr at room temperature. The beads were washed into 0.2 M NaBorate, pH 8.0, and resuspended in 1 mL of borate containing 20-mM DMP.The tube was rotated at RT for 30 min, 250 μL of 1 M Tris, pH 8.0 wasadded, and incubated for 15 min. The beads were washed into PBS+0.05%triton X-100, and resuspended in 1 mL. About 0.4 μL of beads were usedper CANARY assay for most experiments.

Biotin Crosslinking

Antibody was concentrated to ˜1 mg/mL prior to conjugation using Nanosep30K Omega centrifuge concentrators. Biotin (Sulfo-NHS-LC-LC-Biotin,Pierce) was resuspended in PBS to 10 mM. Biotin was added to a 20 foldmolar excess over antibody (equilibrated in PBS) and incubated at RT for30 min. Tris, pH 7.5 was added to 100 mM, and the buffer exchanged intoPBS. Biotinylated antibodies were added to M-280 Dynabeads (Dynal) atsufficient concentration to saturate binding sites (20 μg of antibodyper mg beads) and incubated at RT for 30 min. Beads were collected andwashed and stored in PBS+0.05% Triton X-100. Typically the beads werediluted to one-tenth of their original stock concentration, and 0.4 μLof beads used per CANARY assay.

Introduction

CANARY has demonstrated exceptional performance in the detection of bothviruses and bacteria. Detection of toxins presents a different problem.The difficulty with detecting toxins is that while an antibody expressedon the surface of B cells can bind to two toxin molecules, each toxinmolecule can only bind to one antibody. This means that the antibodieswill not be crosslinked by soluble, monomeric toxin, and consequentlythat the intracellular cascade leading to light emission from the CANARYcell will not be initiated.

An effective method to overcome this problem is to capture toxins onbeads. These toxin-decorated beads can then crosslink multipleantibodies on the surface of CANARY cells and stimulate light emission.The use of capture-beads also facilitates the transfer of solubleprotein toxin from cell-incompatible solutions (containing nonspecificstimulators or inhibitors of CANARY cells) into CANARY cell-compatiblesolutions. This important capability greatly expands the types ofmatrices in which CANARY can potentially be used to detect toxins.

Botulinum Toxin Detection

Toxin Forms: Several types of botulinum neurotoxin A (BoNT/A) antigenwere used, depending on the purpose of the experiment and the maturityof the toxin assay.

GST fusion of BoNT/A heavy chain (BoNT/Hc) produced in E. coli. Thisrecombinant protein was used for screening pools of CANARY cells forthose expressing BoNT/A antibodies. The GST fusion allowed for facileattachment of the antigen to beads and screening of CANARY cells.Recombinant BoNT/E Hc was used as a control to demonstrate thatresponses from CANARY cells were specific to BoNT/A (the antibodies donot bind to BoNT/E). However, GST proteins have a propensity to dimerizein solution, and are therefore not a suitable target to demonstrate theability of CANARY to detect monomeric proteins.

Commercial BoNT/Hc.

This nontoxic portion of BoNT/A is isolated from native toxin and mustbe captured from solution using an antibody against BoNT/A. This is agood model for detection of soluble protein, but the heavy chain portionof BoNT is not as stable as the holotoxin, and this instability madesensitivity measurements using this antigen difficult. Importantly, italso does not actually demonstrate the ability to detect active BoNT/A.

BoNT/A.

Most experiments were carried out using active BoNT/A purchased from acommercial source (Metabiologics).

BoNT/A Complex. BoNT/A as produced by Clostridium botulinum is complexedwith a variety of other proteins. These associated proteins blockbinding of some antibodies, so it is necessary to demonstrate that theCANARY assay developed using these antibodies can detect not only BoNT/Abut also BoNT/A Complex.

Antibodies

Most experiments used antibodies derived from hybridomas 6E10-10, 6C2-4.and 6B2-2. These antibodies bind to independent epitopes on BoNT/A. Mostof the experiments described below used CANARY cells expressing the6B2-2 antibody to detect BoNT/A antigen captured on 6E10-10 antibodybound to beads.

Additional experiments also used antibodies CR1, Raz, and S25, each ofwhich bind to 3 separate epitopes on the BoNT/A protein. Theseantibodies were used to determine the effect of antibody affinity onCANARY assay sensitivity.

Beads:

Glutathione sepharose was used to capture recombinant BoNT/A Hc forpresentation to CANARY cells for screening and initial testing. ProteinG coated beads (sepharose or paramagnetic) were crosslinked to captureantibody and used to capture soluble BoNT/A products in solution forpresentation to CANARY cells. Streptavidin-coated paramagnetic beadswere coated with biotin-labeled antibody. These beads were morereproducible, and because they are paramagnetic, also allow samplepreparation (toxin capture and bead washing) without requiringcentrifugation.

Results: Experiments Using Stimulants for BoNT/A Toxin

The genes encoding antibodies to different epitopes on the BoNT/A Hc(6B2-2 and 6E10-10) were cloned and expressed in separate B cell linesto assess their function. Both resulting cell lines respond to theBoNT/A Hc-GST fusion protein bound to glutathione-sepharose beads. Totest for CANARY cell function, the recombinant antigen was captured onglutathione beads, the beads washed in assay medium, and the captureantigen presented to CANARY cells expressing antibody 6E10-10. The 6B2-2CANARY cell response could be partially abrogated by incubating thebead-bound BoNT/A Hc-GST with 6B2-2 antibody for 2.5 h or overnight.BoNT/E Hc-GST captured on glutathione beads does not stimulate thecells, demonstrating that the CANARY response is stimulated byinteraction with antigen, and not nonspecifically by the beads or thetoxin.

GST proteins dimerize in solution, and therefore cannot be used todemonstrate the ability of CANARY to detect soluble, monomeric protein.To show capture of soluble, monomeric antigen from solution, we usedBoNT/A Hc purified from native BoNT/A (Metabiologics). The 6E10-10antibody was conjugated to protein-G-labeled beads, and these beads wereincubated with different concentrations of BoNT/A Hc. CANARY cells wereadded to the BoNT/A Hc-decorated beads, and the mixture centrifuged for5 sec in order to co-sediment the beads and cells. The captured antigeneffectively stimulated CANARY cells in a dose-specific manner, with anapparent sensitivity of 800 pg (80 ng/ml). The total assay time for thisexperiment was <5 min, including bead binding, cell addition, and lightoutput measurement.

However, BoNT/A Hc aggregated during storage, making accuratemeasurements of assay sensitivity difficult. Unfrozen BoNT/A Hc produceda higher response than BoNT/A Hc that had been frozen. The supernatantof centrifuged, frozen-thawed BoNT/A Hc exhibited even less activity,indicating that aggregates had formed during the freeze-thaw process. Inaddition to the storage characteristics, lot-to-lot variability alsoaffected our ability to accurately determine sensitivity. Since it isimportant to demonstrate that CANARY is capable of detecting solubleprotein, we typically assayed BoNT/A Hc that has been stored frozen, andcentrifuged upon thawing to remove aggregates.

Some solutions, such as orange juice or water, are incompatible with theCANARY assay, so it was necessary to exchange the original solutioncontaining the toxin simulant with assay medium. In addition, somematrices were found to affect not only the cells, but also the captureof toxin by antibody-coated beads. For example, orange juice wasproblematic because of its low pH (pH=3.5). Our solution was to design asingle buffering agent that, when added to a wide variety of solutions,normalized the pH and created some minimal salt concentration to allowspecific capture of antigen. For these experiments, we created aconcentrated buffer (7×HNa) to add to all liquids to raise the salt toat least 80 mM final, and to buffer the pH of acidic solutions likeorange juice to about 6.5. The beads could be stored in this buffer, sothe toxin assay still only required the addition of a single solution(7×HNa+capture beads) to the sample. The antibody-coated beads wereincubated in solution for 12 min, washed with assay medium and used inthe CANARY assay. The LOD for BoNT/A Hc in orange juice and PBS-Tx-100,defined as 3 fold over background, was 80 ng/ml. While the sensitivityof CANARY to BoNT/A Hc in orange juice and PBS/Tx-100 was comparable tothe control, milk proved to be inhibitory (approximately 20% ofcontrol), indicating that the sample preparation would have to bealtered to achieve ideal sensitivity in this matrix. Initial resultsindicate that increasing the salt concentration in milk may improve thesensitivity.

Several medically relevant matrices have also been tested and eachrequired a specific sample preparation method. The procedure developedfor assaying nasal samples had the samples collected on swabs, the stemof the swab was trimmed, and the swab end placed into a 5 micron filterbasket fitted over an eppendorf tube. Assay medium containing BoNT/A Hcwas added, and the assembly capped and centrifuged. The filtered eluatewas collected in the eppendorf tube and assayed using the bead-captureprocedure described above. The signals from actual and mock swabs withBoNT/Hc are very similar, indicating that no inhibitors are present inthe nasal sample. The lack of a CANARY response to nasal swabs in theabsence of antigen (nasal swab) shows that there are no nonspecificstimulators present in the nasal swab sample.

BoNT/A Toxin and CANARY Assay Sensitivity

To demonstrate that CANARY detects not only with BoNT/Hc toxin simulant,but also the active BoNT/A toxin, commercial BoNT/A was acquired andassayed using toxin captured with 6E10-10 beads and detected using 6B2-2CANARY cells. The limit of detection in this assay was about 8 ng/ml or80 pg of the toxin, which is an improvement of approximately 10 foldbetter than the LOD for BoNT/A Hc toxin simulant. Samples containing 16pg of toxin (1.6 ng/ml) stimulate cells to about 3 fold over background,but with a kinetic profile that does not fit the current detectionalgorithm. This improvement in assay sensitivity indicates either thatthe active BoNT/A toxin remains soluble during storage, or that theantibodies bound better to the whole toxin than to the heavy chain.

Detection of BoNT/A toxin in actual samples was also demonstrated. Thedetection of BoNT/A toxin in urine was performed where the limit ofdetection was 16 ng/ml CANARY was also effective for the detection ofBoNT/A in whole blood. BoNT/A was added to whole blood, and the bloodbriefly centrifuged through a polymer to facilitate separation of cellsfrom soluble material. 6E10-10 beads were added to the resultingsupernatant, incubated for 2 min, and assayed using 6B2-2 CANARY cells.As was observed when detecting toxin simulants in milk, the limit ofdetection for this assay, 16 ng/mL, is about 5 fold lower than thesensitivity seen using control medium.

It is possible that the high protein concentration in both of thesematrices inhibited specific interactions between the bead-boundantibodies and the BoNT/A in solution. In an effort to improve thesensitivity in high protein solutions, the addition of salt and nonionicdetergents was tested. Salt (NaCl), nonionic detergent (Tween-20 orTriton X-100) or combinations of the two were added to 48-ng/ml BoNT/Ain plasma, and the results compared to the addition of water. Theaddition of Triton X-100 improved the signal, while addition of Tweendid not. Addition of salt alone had a more dramatic effect, increasingthe amplitude of the signal from 1700 RLU to about 4800 RLU. Addition ofdetergent to samples containing salt did not produce an additive effect.This indicates that addition of salt may have decreased nonspecificprotein-protein interactions and increased the rate of BoNT/A binding tothe antibody-coated beads.

Assay Optimization

The sensitivity of the BoNT/A assay would be expected to be dependent onthe density of antigen on each bead which, in turn, is dependent on thenumber of beads used to capture the toxin in solution. Using a largenumber of beads ensures the maximum capture efficiency, but if theconcentration of toxin is low the antigen present on each bead may betoo sparse to elicit an effective cellular response. Therefore, abalance between bead number and antigen density on each bead must bestruck. In order to optimize these parameters, a set of experiments wasperformed testing a variety of bead concentrations with differentvolumes of BoNT/A at 1.6 ng/mL. In one such experiment, differentnumbers of beads were added to each sample and were incubated for 2 min.When incubated in small volumes, large numbers of beads stimulated thecells less well than small numbers of beads. This would indicate that insamples containing low amounts of toxin, capturing with large numbers ofbeads results in too sparse a distribution of antigen to effectivelystimulate the CANARY cells.

While extending the capture time significantly improves the LOD to 0.32ng/ml of BoNT/A, we also observed that the effects of bead number becamemore pronounced. For example, with beads incubated overnight in 100 μlof BoNT/A at 0.32 ng/ml, decreasing the number of beads from 300,000 to3,000 improved the signal. Fewer beads means each bead will have moretoxin, improving the signal as the number of beads decreases.

The combination of biotinylating the antibody, improving binding andwashing conditions, and optimizing bead number led to improvedsensitivity of 16 pg (1.6 ng/ml) in a 6-min assay. Sixteen picograms oftoxin represents about 0.000029 (1/34,370) of the LD50 by inhalation fora 55-kg (120 lb) person. This is about 0.00023×LD50 by injection, and0.00000029×LD50 by ingestion. At this level of sensitivity the assaycould detect about 1 LD50 present in 34 liters of fluid.

Results for Real Toxin: BoNT/A

BoNT/A spiked into urine could be detected, although the signalamplitude was somewhat reduced compared to controls. (FIG. 158) In thisexperiment no pretreatment was used, and the 6E10-10 coated beads wereadded directly to urine spiked with BoNT/A. The limit of detection forBoNT/A in urine was 16 ng/ml, compared to 3.2 ng/ml for toxin diluteddirectly into assay medium in parallel experiments.

CANARY was also effective for BoNT/A screening in whole blood using thesample preparation procedure described elsewhere (FIG. 159). Whole bloodwas spiked with BoNT/A, and the blood briefly centrifuged through apolymer to facilitate separation of cells from soluble material. 6E10-10antibody-coated beads were added to the resulting supernatant, incubatedfor 2 minutes, and assayed using 6B2-2 CANARY cells. The limit ofdetection for this assay is 16 ng/ml, about 5 fold lower than thesensitivity seen using control medium. This sample preparation methodresulted in a significant improvement over previous assays on plasmaprepared by centrifugation in the absence of polymer.

In both milk and serum, the limit of detection for toxin by the CANARYassay was about 5-fold higher than controls. It is possible that thiswas because the high protein concentration in both of these matricesinhibited specific interactions between the bead-bound antibodies andthe BoNT/A in solution. In an effort to improve the sensitivity in highprotein solutions, the addition of salt and nonionic detergents wastested (FIG. 160). Salt (NaCl), nonionic detergent (Tween-20 or TritonX-100) or combinations of the two were added to 48 ng/ml BoNT/A inplasma, and the results compared to the addition of water. The additionof Triton X-100 improved the signal, while addition of Tween did not.Addition of salt alone had a more dramatic effect, increasing theamplitude of the signal from 1700 RLU to about 4800 RLU. Addition ofdetergent to samples containing salt did not produce an additive effect.This indicates that addition of salt may have decreased nonspecificprotein-protein interactions, and increased the rate of BoNT/A bindingto the antibody-coated beads.

We have shown that CANARY can effectively detect active BoNT/A, but ifthe toxin is isolated from certain strains of Clostridium botulinum, thetoxin will be complexed with additional proteins, creating anantigenically different target, BoNT/A Complex. Importantly, CANARYdetected BoNT/A Complex with the same response levels as BoNT/A (FIG.161). Equimolar amounts of BoNT/A and BoNT/A Complex (5 fmoles of each)were added to 6E10-10 coated beads, and the captured toxin detectedusing 6B2-2 cells. The CANARY response was identical to bothpreparations, indicating that the epitopes on the BoNT/A bound by theseantibodies were not blocked by the BoNT/A Complex proteins.

We have chosen to focus on developing an assay that is very fast. Longerincubations are of interest in determining the limits of the assay, butnot for diagnostic or detection purposes. We found that biotinylatingthe capture antibody and attaching it to streptavidin beads was easierand gave marginally better results. The combination of biotinylating theantibody, improving binding and washing conditions, and optimizing beadnumber led to improved sensitivity over a period of time. In an assay on10 μl of suspect solution spiked with BoNT/A, the sensitivity of the is16 pg (1.6 ng/ml) (FIG. 162). The entire assay, including addition ofthe beads, binding for 2 minutes, magnetic capture and bead washing,cell addition and light output measurement takes about 6 minutes.

Summary

In summary, we have developed an assay for Botulinum toxin usingantibody-coated beads to capture soluble toxin. These toxin-decoratedbeads are used to present immobilized toxin to CANARY cells.Importantly, the beads also facilitate the transfer of toxin from anyvariety of cell-incompatible matrices into assay media. This allowsdetection of toxin in blood, urine, nasal swabs, orange juice, milk,water, and PBS-Triton. Some matrices cause decreased responses in theCANARY assay, particularly those that contain high concentrations ofprotein (plasma and milk). This inhibition can be partially overcome byadding salt to decrease nonspecific protein interactions. The assay hasbeen optimized for speed, and can detect 16 pg (1.6 ng/ml) BoNT/A in 6min. Sensitivity would seem to be dependent on the affinity of thecapture antibody, but the use of higher-affinity antibodies does notimprove the limit of detection. Increasing the incubation time of thebead-capture step does result in better sensitivity (less than 0.32ng/ml. Even in difficult matrices the assay can detect a fraction of anLD50 in 6 min.

Hardware Development for CANARY

Materials and Methods

Magnetic Agent Bead and Magnetic B-Cell Bead Assay

B-cell binding beads: Dynabeads® Mouse Pan B (B220) Catalog Number114-41D were used without further modification.

Agent-binding beads: Dynabeads® M-280 Tosylactivated Catalog Number142-03 were functionalized with capture antibodies according to themanufacturer's recommendations.

Assay procedure: Incubate magnetic beads (Dynal/cat. no. 142-03) coatedwith agent antibodies in 1.5-ml tube with sample for 5 min at roomtemperature. Pull captured agent and bead down to bottom of tube with amagnet. Add B-cell magnetic beads (Dynal/cat. no. 114-41D) to tube andpull them down to bottom of tube using a 10-sec exposure to a strongrare-earth magnet. Place tube in a luminometer and read signal.

Lateral Flow Strips

Materials:

Sample pads: Millipore glass fiber pads G041/GFCP1 030 00. Wick pads:Millipore cellulose absorbent sample pads C082/CFSP1 730 00

Capture membrane: Pall 0.45-μm GH polypro membrane (cat. no.GHP4550001/Pall)

Methods

Assemble the lateral flow strips as follows. Place a 0.25-in.×0.25-in.Millipore wick pad onto packing tape. Apply 0.4-in.×0.1-in. Pall 0.45-μmGH polypro membrane on top of wick pad so that ⅓ of membrane is on topof wick pad. Apply 0.25-in.×0.5-in. glass fiber filter to Pall GHpolypro membrane.

Single-Channel Sensor Development

Described herein are improved single-channel hardware capable ofperforming optimal CANARY assays. We pursued two parallel paths: (1)Development of custom design concepts for a single unit capable ofspinning and analyzing the CANARY samples, and (2) examining COTSluminometers and minicentrifuges that could be modified, or preferablyused without modification, to perform single CANARY assays. The outcomeof that process was the identification of inexpensive COTS hardware thatimproved CANARY assay procedures and performance. The optimum hardwarecombination consisted of the Berthold Detection Systems FB12 luminometerused in conjunction with a VWR minicentrifuge fitted with a custom rotorto enable spinning of up to eight CANARY samples in the optimumconfiguration.

The procedure for using the single-channel sensor begins with a ˜2-minpre-spin at >6000 RCF in a conventional swing-bucket microcentrifuge, ifavailable, or in the VWR minicentrifuge. A drop of B cells was added tothe sample, placed in the minicentrifuge and spun for 5 sec. There issufficient time before the signal peaks to transfer the sample to theluminometer for signal readout and CANARY identification. The entireCANARY test procedure can be completed in 3 min enabling thissingle-channel CANARY sensor operated by a single user to process up to25 samples per hour with parallel sample pre-spins.

16-Channel Sensor Development

In its simplest form, a CANARY measurement consists of preparing asample in a transparent tube, introducing an aliquot of speciallyprepared B cells into the tube, driving the B cells to the bottom of thetube using a quick centrifugal spin, and measuring the light output fromthe tube with a photon-counting sensor. In the laboratory, most CANARYmeasurements have been made sequentially, one sample at a time; in theautomated BAWS/CANARY bioaerosol identification sensor, four samples aremeasured simultaneously, each sample having its own light-gatheringchannel. Each light-gathering channel typically consists of a photonsensor, high-voltage power supply, a pulse-discrimination circuit, andpossibly a digital counter. The former system requires more time, whilethe latter requires more complex (and expensive) hardware.

A new approach that reduced the time to measure multiple samples (whilekeeping the hardware requirements minimal) was successfully tested. Asensor testbed was fabricated that allows the simultaneous measurementof up to 16 samples using a single light-gathering channel. The sensorconsisted of a rotor holding 16 1.5-ml tubes horizontally, equallydistributed about its circumference, and driven by a variable speedmotor about a vertical axis. A single fixed photon-detecting element (inthis case, a PMT) was positioned in the plane of the rotor just beyondthe path of the tubes during rotation. In this way, each of the tubeswas sequentially and repetitively brought into close proximity to thePMT, allowing its light output to be sampled on each pass.

Additionally, an optical switch consisting of an optical source (aninfrared LED) and a detector (a phototransistor) was used to control thecounting of detected photons and the reorganization of the data into 16fields, each associated with a specific sample.

A single measurement consists of: 1. Preparing 16 samples (and/orcontrols) in individual 1.5-ml tubes; 2. Introducing an aliquot of Bcells into each of the tubes; 3. Installing the tubes into the rotorsituated in a dark box; 4. Localizing the B cells at the bottom of thetubes using a brief (5 sec) centrifugal spin at high RCF (˜2000 g); 5.Reducing the rotor speed to 60 rpm for the duration of the measurement(1-2 min), each tube being sampled once every second; 6. Generating atime series of photon counts for each sample for display and/or input toa computer algorithm for evaluation.

Tests were run analyzing the signal collection characteristics fromassays read while spinning to determine how fully the 16-place rotor inthe testbed could be populated before signals began to overlap. With therotor fully populated, all of the samples produced signals with signalto noise ratios comparable to those observed in the single-channelsensor, and no observable crosstalk of emitted light between channelswas observed if sufficient baffling was provided to limit thetransmitted angles for the light. An example of the data from the16-channel testbed shows an LOD comparable to that of the single-tubemethod. While this sensor measures 16 samples as designed, larger samplenumbers are possible, though physical size and the statistics ofsampling will ultimately dictate practical limits.

The rotary format was incorporated into the design for the portable16-channel prototype sensor. The primary goal of the design was toincorporate the hardware necessary for spinning and readout of CANARYassays into a small, self-contained portable unit less than 12 inches inthe longest dimension. Additionally, provision was made to ensure thatpower consumption was low enough to enable inclusion of a battery intothe enclosure for battery-powered operation. These goals wereaccomplished by building the sensor components into a small COTStransportation case that was water and light tight, and by using asmaller motor and controller that was capable of spinning the rotorusing a 24-V DC power source.

Handheld Sensor Development: Simplified Assay Development

A compact handheld sensor targeted at clinical, point-of-care, andforward-deployed applications is of particular interest. We have focusedon characterizing the performance of alternative assay procedures thatcan reduce or eliminate the requirement for centrifugation steps sincethey are currently the primary driver of energy consumption andinstrument complexity. We experimentally evaluated a number ofapproaches toward assay formats that employ reduced centrifugationrequirements, microfluidic channels, lateral-flow assemblies,filtration, or magnetic bead capture. Of these approaches, reduction ofthe centrifugation requirements, use of lateral-flow assemblies, andmagnetic bead capture are described in more detail below.

Standard format with reduced centrifugation steps. Signals in responseto high concentrations of agent have been observed withoutcentrifugation steps, so in order to characterize the performancetradeoffs that would result, we performed a series of experiments usingdifferent centrifugation permutations. Experiments indicate thatreducing the centrifugation and assay times (from ˜3 min per assay to ˜1min per assay) will reduce the sensitivity by approximately one order ofmagnitude.

Lateral-Flow Formats.

We have characterized CANARY assay performance in devices that layerwicking and filter materials to accomplish sample fluid transport andantigen localization without centrifugation. The basic construction ofthe device as well as pictures demonstrating its ability to localizespore-sized particles are shown in FIGS. 184 and 185. FIG. 186 shows theresulting CANARY signals for both standard centrifuge assays andlateral-flow assays using the same agent and cell samples.

Dual-Magnetic-Bead Assay.

We have characterized an assay that takes advantage of two sets ofmagnetic beads. One set is specific for the CANARY B cells, while theother set is specific for a particular agent. In FIG. 187, a standardCANARY assay was run alongside a dual-bead assay using the same B cellsand agent dilution series. Magnetic beads specific for Y. pestis weremixed with a dilution series of Y. pestis agent for 5 min. After 5 minthe magnetic beads were pulled to the bottom of the assay tube alongwith any bound Y. pestis, and the supernatant was removed. Magneticallylabeled B cells were then added to the sample and pulled down to thebottom of the tube. Localizing agent and B cells with magnetic beads hasthus far proven to provide similar sensitivity to that ofcentrifugation.

Handheld Sensor Hardware Development

Handheld sensor hardware development began with the design of acartridge capable of a single CANARY assay that can be performed withoutcentrifugation. The cartridge was designed to contain a swab that has asmall but powerful magnet in its tip, as well as a capsule of B cellsthat are attached to magnetic beads (FIG. 188). After using the swab tosample a surface, it would be introduced to the capsule containing the Bcells, and the magnet would draw the bead-bound cells to the antigen onthe surface of the swab. Then the entire cartridge would be slid into aspecially adapted, battery-powered luminometer to record the lightemission. This handheld sensor could be used in the field to determineexposure of a person or surface to a specific pathogen. The rationalefor this design was based on several factors. We have demonstrated theability to replace centrifugation with magnetic beads by using a magnetto draw bead-bound B cells to the antigen in a liquid sample (that wasprepared by centrifugation). We have also shown that B cells can bepackaged in capsules, as they would be in the cartridge, and eitherrefrigerated for weeks or held at room temperature for 48 h withoutlosing sensitivity. Finally, although magnets can have adverse effectson the function of a photomultiplier tube, we have been able to showthat the distance between the magnetic swab and the photomultiplier tubein the luminometer can be controlled to prevent these adverse effects.Initial experiments have shown that bead-bound B cells, drawn to aspherical neodymium magnet in the absence of antigen, give off atransient light signal. This is most likely due to mechanical stress onthe cells. Several possible remedies were identified including: use ofweaker magnets (neodymium magnets are very strong); “tuned” magnets (amagnetic material at the tip of the swab, magnetized by a neodymiummagnet mounted further away in the body of the swab); and a retractablemagnet (which can be withdrawn immediately after the B cells have beenattracted to the swab surface).

The complexity of magnetic manipulation and processing was removed fromthe consumable where it would drive up the cost of operation. Shiftingthe components required for magnetic sample and cell manipulations intothe handheld readout device adds little to the overall cost of thedevice. Furthermore this approach enables the assays to be performed inCOTS microcentrifuge tubes and ensures maximum sensitivity andreliability. Based on these advantages, the a handheld luminometer withfeatures enabling onboard magnetic assay manipulation was developed. Theoptical sensor and supporting electronics are based on those found in acommercially available luminometer made by Berthold Detection Systems,the same manufacturer that produces the COTS luminometer that wasincorporated into the single-channel CANARY sensor. The design thatresulted is shown in FIG. 189. The completed sensor based on this designis shown in FIG. 190.

The handheld CANARY sensor (FIG. 190) features a PMT oriented with thephotocathode<1 mm from the bottom of the assay tube, a readout screenwith touch pad, a rechargeable battery pack, and a sliding sample door.The sample door contains a rare-earth magnet positioned so thatinsertion of a tube as shown in FIG. 189 results in co-localization ofthe captured target and magnetically labeled B cells. The assayprocedure begins with the addition of magnetic beads to the sample,followed by mixing and incubation for 5 min. The sample/bead suspensionis then placed in the magnetic tube holder on the door for 1 min tolocalize the captured target at the bottom of the tube. The sample thathas been depleted of target is removed and replaced with assay buffercontaining B cells, and the tube is returned to the magnetic holder.After 5 sec the tube is placed into the read position on the door, thedoor is closed, and the PMT signals are recorded.

Thus, we have developed a system for producing genetically engineered Bcells that serve as sensors for the rapid identification of pathogensand toxins. The assays we have developed using these cells demonstratethe best combination of speed and sensitivity known (<50 particles ofkilled Y. pestis in <3 min, with a false-alarm rate of 0.4% withlaboratory samples), and because the B cells are self-replicating, thecost of the materials is very low. In addition to the 24 geneticallyengineered B-cell lines we have generated, including Rift Valley Fever,Dengue viruses, and others of significance to clinical diagnostics, wehave produced a CANARY cell line whose specificity can be engineered indays instead of months. We have developed 5-min assays for clinicallyrelevant samples, demonstrating detection of 50 cfu of B. anthracisspores from nasal swabs, 500 C. trachomatis EBs in urine, and 1000 cfuof Y. pestis/mL of whole blood. We have also demonstrated that CANARYassays can be multiplexed by combining up to three cell lines in asingle assay, or by engineering cells that respond to more than onepathogen. Alternatively, we have shown the production of B cells thatemit different wavelengths of light, enabling a single assay that candistinguish between two or more pathogens.

We have extended the capabilities of CANARY to include protein toxins,demonstrating detection of as little as 16 pg (1.6 ng/ml) of Botulinumtoxin A in a 6-min assay. Sixteen picograms of toxin represents about0.000029 (1/34,370) of the LD50 by inhalation for a 55-kg (120 lb)person. This is about 0.00023×LD50 by injection, and 0.00000029×LD50 byingestion. At this level of sensitivity the assay could detect about 1LD50 present in 34 liters of fluid. It is unclear whether thissensitivity would be sufficient for diagnosis of BoNT/A using serumsamples from patients (published data on serum concentrations arelacking), but it would certainly be an excellent screening method forfood contamination, aerosolized material, or inhalation exposure (nasalswabs).

Although the CANARY assay can be performed in a single-channel formatusing several pieces of COTS equipment, we have developed a 16-channelsensor with an integrated spin motor and PMT that can processapproximately 100 samples/hour while maintaining the optimum LOD of 50cfu/pfu of bacteria or large viruses. We have also developed a handheldsensor that utilizes a noncentrifugal, dual-magnetic approach.

The CANARY B-cell-based biosensor exploits a highly evolved system forpathogen identification that provides several advantages over otheridentification technologies. With CANARY it is possible to provideidentification in approximately 5 min, including sample preparation, andwith those pathogens large enough to be concentrated in a microfuge, wehave demonstrated a level of sensitivity that approaches PCR. Incomparison, state-of-the-art immunoassays require at least 14 min andhave a higher limit of detection (6×□10⁴ cfu or 6×□10⁶ pfu). While PCRis extremely sensitive (1 to 5 cfu), highly specific, and has enjoyedtechnological breakthroughs that have reduced the time for amplificationand signal detection, the assay takes at least 7 min (typically 20-30min), not including the time required to extract and purify the DNA.Applications that would benefit from a technology such as CANARY includepoint-of-care diagnostics for illnesses where the return rate fortreatment is low but the societal impact is high, such as sexuallytransmitted diseases. In addition, CANARY would be valuable forpre-symptomatic detection from nasal swabs in the aftermath of abiowarfare attack, detection of agricultural pathogens at ports ofentry, or screening of perishable food supplies. In fact, CANARY is arapid, sensitive method that can enable the detection and identificationof highly infectious pathogens in any time-critical setting.

Cell Engineering and Assay Method Examples A. Cell Engineering Methods

M12g3R cells were maintained at 37° C. in a humidified atmosphere of 5%CO₂ in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodiumpyruvate, 2 mM L-glutamine, 100 μM nonessential amino acids, 50 μM2-mercaptoethanol, 50 μg/ml streptomycin, and 50 U/ml penicillin, 250ng/ml amphotericin B (Life Technologies). Cells were transfected withpCMV.AEQ.IRES.NEO via electroporation (270 V, 950 μF) and selected in 1mg/ml G418 for two weeks. G418-resistant clones were screened forresponse to anti-IgM. Those clones with the greatest increase in photonemission upon crosslinking of the surface IgM were used in subsequenttransfections to generate B cell lines specific for particularpathogens. Surface expression of antibodies with engineered specificityis accomplished by co-transfection (via electroporation) with expressionvectors for light and heavy chains, as well as with one that encodes agene conferring resistance to puromycin. Puromycin-resistant pools andclones were selected on the basis of their response to antigen. Thelight chain expression vector, VKExpress, contains the constant regionfor the human kappa gene downstream of a multiple cloning site (MCS),under control of the human elongation factor-1α (EF-1α) promoter. Theheavy chain vector was generated by modifying pDisplay (Invitrogen),retaining the cytomegalovirus (CMV) promoter and leader sequence, butreplacing the platelet-derived growth factor (PDGF) receptortransmembrane domain with the gene for the membrane-bound constantregion of murine IgM and removing both tags on either side of the MCS.The appropriate restriction sites are added to the antibody variableregions using PCR and the sequence of all PCR products is confirmedbefore cloning into the expression construct. The variable regions usedto produce the recombinant antibody were cloned either from cDNA or fromhybridomas using Reverse-Transcription (RT) with random oligonucleotideprimers and PCR. RNA was extracted with Trizol reagent (LifeTechnologies), according to the manufacturers recommendations, and firststrand synthesis performed using the Retroscript kit (Ambion). PCR wasaccomplished using sets of primers designed to anneal to the leadersequences of either light or heavy chains [S. T. Jones and M. M. Bendig,Bio/Technology 9, 88 (1991)] at the 5′ end, and the constant regions ofmurine Kappa or IgG2 at the 3′ end.

B. Bioluminescent B Cell Response to FMDV

The M12g3R B cell line, stably transfected with the pCMV.AEQ.IRES.NEOplasmid and expression vectors for a recombinant antibody thatrecognizes the A12 strain of FMDV, was prepared for the luminesenceassay as follows: Cells were thawed on Day 1. Preparation of the cells24 hours post-thaw is critical for maximum activity and reliability. Thefreeze/thaw step increases the response of the B cells by as much as 100fold. On Day 2, 10⁶ cells were incubated at room temperature for 2 hoursin assay medium [CO₂—Independent medium, 10% FBS, 50-μg/ml streptomycin,and 50-U/mil penicillin, 250 ng/ml amphotericin B (Life Technologies)]with 50-μM coelenterazine (Molecular Probes, Eugene, Oreg.) covered withfoil, washed twice, and resuspended in assay medium at a finalconcentration of 5×10⁵ cells/ml. Cells were left rotating overnight atroom temperature in 1.5 ml microcentrifuge tubes and assayed 15-20 hourslater. For the assay, 25 μl of cells was mixed with antigen (5 μl of thewt A12pRMC35 strain at 1.4×10⁸ pfu/ml, 10 μl of the A12 variant, B2PD.3,at 7.5×10⁷ pfu/ml) and the response measured in a luminometer (Lumat LB9507, Perkin Elmer).

C. Bioluminescent Assay with Bacteria and Large Viruses

The sensor device and methods can be used for the rapid detection ofbacterial, as well as viral pathogens. Cell lines were engineered torespond to the bacterium, Francisella tularensis, the etiological agentof tularemia. However, when assayed using the same protocol as with theFMD and VEE viruses, the signal is slow and almost indistinguishablefrom background, indicative of low interaction rates between the B cellsand antigen (0s pre-spin/0s spin). Previous experiments performed withantigen-bead simulants have indicated that sensitivity and speed couldbe augmented by concentration of antigen and B cells (data not shown),so the luminometer was re-configured to include a centrifuge positionedabove the photomultiplier tube (PMT). When the agent and cells are mixedtogether, then concentrated by centrifugation for 5 seconds, the signalis improved and the response faster (0s pre-spin/5s spin). Optimalresults are observed when the slower-sedimenting F. tularensis iscentrifuged prior to the addition of the cells (60s pre-spin/5s spin).This format ensures that a large number of cells come into physicalcontact with antigen within a short time frame, thereby providing amajor improvement in sensitivity and speed. After additionaloptimization of the assay protocol, we can now detect as little as 60colony-forming units (cfu) of F. tularensis in less than 3 minutes,including the time it takes to pre-spin the agent, but see no responseto inactivated Yersinia pestis, the bacterium that causes the plague.This limit of detection has been confirmed with two other sources ofinactivated F. tularensis, and one different strain (data not shown).Furthermore, the sensor device exhibits a wide range of sensitivity,detecting concentrations ranging over 7 orders of magnitude.

B cells were prepared as described above. 50 μl containing the indicatedamounts of Y. pestis or F. tularensis were centrifuged for 60 s at6500×g, then 20 μl of cells were added and spun an additional 5 s in thecentrifuge luminometer. Photons were detected with a Hamamatsu HC-125photomultiplier tube and the signal monitored with a Stanford ResearchSystems SR400Gated Photon Counter.

Nucleic Acid Detection Example Characterization of Emittor CellsExpressing Digoxigenin Antibody

Plasmids encoding an antibody (Daugherty et al. (1998) ProteinEngineering 11 (9): 825-832) against digoxigenin were introduced intoemittor cells, and these cells were screened using protein (BSA)chemically conjugated to digoxigenin (Dig-BSA). Twelve independent poolswere selected resulting in 12-24 independent cell lines. The firstexperiment tested whether these cells could detect digoxigenin antigenscrosslinked by DNA (Dig-DNA). Three types of commercial Dig-DNA havebeen tested for reactivity with Dig antibody expressing CANARY cells(FIGS. 26A-C): plasmid DNA with a digoxigenin attached every 20 basepairs (FIG. 26A); DNA molecular-weight markers with digoxigenin attachedevery 200 bases (FIG. 26B); and DNA molecular-weight markers with onedigoxigenin attached to each end (FIG. 26C). Each of these standardsstimulated the emittor cells to a varying degree, with the mostsensitive response being to the Dig-labeled plasmid DNA. The fact thatantigens spaced an average of 20 bases apart stimulate the cells 100fold more (on a per digoxigenin basis, not on a per microgram of DNAbasis) than antigens spaced 200 bases apart is an indication that 200bases is too great of a distance to stimulate an ideal response. Inorder to stimulate an intracellular cascade resulting in calcium releaseand aequorin light production, adjacent antibodies must be immobilizednear enough to each other to initiate the reaction inside the cell.

It was also noted that centrifugation just before measurement of lightoutput, which is routine in the detection of both soluble and insolubleantigens using traditional CANARY, may actually decrease the sensitivityof CANARY against the soluble Dig-DNA antigen. In the experiment shown(FIGS. 27A and 27B), centrifuging the cells through the DNA solutionappears to decrease the limit of detection by nearly a factor of 10.This result may reflect differences between detection of DNA anddetection of other nonsedimentable antigens.

Detection of Hybridized Oligonucleotide Probes Using Emittor Cells

This assay was designed to detect hybridization of digoxigenin-labeled(Dig-labeled) probes to target DNA. The target DNA for these experimentswas derived from the phagemid pBluescript II. This 3100 base pair-longcircular phagemid can be induced to make double-stranded DNA (dsDNA) oreither of the two single strands of DNA (ssDNA). These two ssDNA strandsare termed the (+) strand or the (−) strand. Ten Dig-labeledoligonucleotide probes that bind specifically to the (+) strand weredesigned:

Phagemid Oligo base number DNA Sequence position Tm  1 GCAACGTTGTTGCCATT2269-2285 56.0 (SEQ ID NO: 1)  2 TACAGGCATCGTGGTGT 2288-2304 53.3(SEQ ID NO: 2)  3 GCTCGTCGTTTGGTATGG 2309-2326 57.3 (SEQ ID NO: 3)  4TCATTCAGCTCCGGTTC 2328-2344 55.0 (SEQ ID NO: 4)  5 ACGATCAAGGCGAGTTAC2348-2365 53.1 (SEQ ID NO: 5)  6 GATCCCCCATGTTGTGC 2368-2384 57.7(SEQ ID NO: 6)  7 AAAGCGGTTAGCTCCTTC 2388-2405 54.3 (SEQ ID NO: 7)  8TCCTCCGATCGTTGTCA 2408-2424 56.5 (SEQ ID NO: 8)  9 GTAAGTTGGCCGCAGTG2428-2444 55.7 (SEQ ID NO: 9) 10 TCACTCATGGTTATGGCA 2448-2465 53.5(SEQ ID NO: 10) NEG3 CCATACCAAACGACGAGC 2326-2309 57.3 (SEQ ID NO: 11)Oligonucleotides are numbered in the order of their location along thepBluescript phagemid DNA. Shown for each is the DNA sequence of theoligonucleotide, the position of that sequence on the phagemid, and themelting temperature (Tm) of that oligonucleotide (an approximation ofthe binding affinity). The small range in Tm's for theseoligonucleotides indicate that they each have similar bindingcharacteristics.

Each of these oligonucleotides has a digoxigenin (Dig) molecule attachedto the first residue, and each have comparable target DNA bindingcharacteristics as reflected by their similar calculated meltingtemperatures (Tm). Hybridization of this set of 10-digoxigenin-labeledoligonucleotides to the (+) strand of the target DNA yields a 200 basestretch of double-stranded DNA with one Dig molecule every 20 bases. Theremaining 2900 bases of the plasmid remain single stranded. Thiscollection of immobilized digoxigenin antigens crosslink digoxigeninantibodies on the surface of emittor cells and stimulate lightproduction.

The 11th oligonucleotide (NEG 3) is a control. NEG 3 was designed tobind directly to oligonucleotide number 3, producing a short piece ofdsDNA 20 nucleotides long with a single Dig on each end. Emittor cellsexpressing a digoxigenin antibody were capable of detecting 80femptomoles of input oligonucleotide (FIG. 28). This controldemonstrated that the hybridization conditions chosen were at leastsufficient to support binding of two oligonucleotides within this Tmrange. More importantly, this control demonstrated that the bindingbetween 20 bases of complementary DNA is sufficiently strong tocrosslink antibodies and elicit a signal from the emittor cell.

Oligonucleotide-oligonucleotide hybridization occurs extremely quickly(FIG. 29). Oligonucleotide NEG3 was added to hybridization solution,followed by Oligo3. The solution was immediately diluted in medium, theemittor cells added, and the reaction place in the luminometer (elapsedtime from addition of oligo 3 was 15 seconds). This abbreviatedhybridization protocol did not drastically affect the sensitivity of theassay (compare FIG. 29 to FIG. 28).

Next, multiple Dig-labeled oligonucleotides were hybridized tosingle-stranded DNA target. This complex was tested for its ability tostimulate emittor cells. FIG. 30 shows a series of hybridizations ofdifferent concentrations of the Dig-oligonucleotide probe set to a givenamount of ssDNA. The ratio of ssDNA:oligonucleotide probe giving thebest signal in this experiment was between 1:2 and 1:4. At higherconcentrations of probe, the unbound Dig-labeled oligonucleotideappeared to inhibit signaling. In these experiments 0.63 pmoles ofoligonucleotide worked well under many of the conditions tested. Adose-response curve gives a limit of detection for single stranded DNAof approximately 50 ng, or about 50 fmoles (FIG. 31). It is important tonote that (−) strand DNA was not detected in either of theseexperiments, indicating hybridization of the Dig-labeledoligonucleotides and subsequent signaling from the emittor cells isdependent on the sequence of the target DNA.

Temperature and buffer constituents affect hybridization of Dig-oligosto target NA. Hybridization at between 47° C. and 51° C. in either PBS(FIG. 32A) or TBS (FIG. 32B) gave the highest response. Hybridizationsperformed at higher or lower temperatures decreases the amplitude of thesignal. Changes in the buffer constituents will obviously affect theideal hybridization temperature.

Target DNA Capture

Biotin-labeled oligonucleotides have been bound to the surface ofstreptavidin-coated magnetic and nonmagnetic beads. These “capture”oligos are designed to bind to the target DNA in a position well removedfrom the location of the Dig-labeled oligonucleotides. Binding thetarget NA to a sedimentable support will allow for more extensivewashing of the DNA before addition of emittor cells, and improve thesensitivity of the assay. One strategy for sedimentation of target NA isshown in FIG. 33. In this scheme, a biotin-labeled captureoligonucleotide is attached either streptavidin-coated polystyrene ormagnetic beads. Digoxigenin-labeled oligonucleotides are hybridized tothe target, and the complex sedimented by centrifugation or applicationof a magnetic field. The emittor cells are then resuspended andsedimented with the beads, and the reaction tube placed in aluminometer. The effects of sedimentation on detection of target DNA isshown in FIG. 34. In this case, the LOD is improved to the high attomolerange as compared to typical results in which the DNA is not sedimented.The addition of a commercial blocking reagent (Roche Applied ScienceCat. #1 096 176) improves signal further. FIG. 35 shows the result ofaddition of different concentrations of blocking agent during thehybridization/capture step. In this experiment, addition of between 1%and 10% blocking reagent improved the signal to background ratio at allconcentrations of target tested.

Fc Receptor Emittor Cells

The Fc receptors are a family of membrane-expressed proteins that bindto antibodies or immune complexes. They are expressed on severalhematopoietic cells including monocytes and macrophages. Severalsubclasses of Fc receptors exist including Fc gamma Receptor I (FcγRI),a high-affinity binder of soluble antibody. FcγRI binds to the constantregion (Fc portion) of Immunoglobulin G (IgG) leaving theantigen-binding region of the antibody free. Crosslinking of theantibody-bound receptor by specific antigen initiates a signalingpathway that stimulates calcium release.

The human macrophage cell line, U937, contains endogenous FCγR1.Treatment of these cells with IFNγ increases the expression of theFcγRI, as seen in FIG. 36A. U937 cells transfected with the aequorinexpression plasmid produce functional aequorin as demonstrated bytreating these cells with the calcium ionophore ionomycin. This causes arapid and transient rise in calcium that stimulates the aequorin to emitlight, as seen in FIG. 36B. U937 cells were then tested to determine ifthe aequorin would be stimulated by the calcium rise initiated bycrosslinking of the Fc receptors. U937 cells were incubated with humanIgG for 15 min at room temperature. The cells were washed to removeunbound IgG and treated with goat anti-human IgG. A rapid rise incalcium was observed, as shown in FIG. 36C.

Experiments demonstrated that U937 cells can be “engineered” rapidly torespond to several different pathogens or simulants. U937 cells weretreated for 24 h with IFN (200 ng/ml) to increase expression ofendogenous FcγRI, and prepared for the emittor cell assay. The cellswere incubated with the following antibodies: mouse anti-B. anthracisspore (FIG. 37A), rabbit polyclonal anti-B. anthracis spore (FIG. 37B),mouse anti-F. tularensis (FIG. 37C), or mouse anti-B. subtilis (FIG.37D). Cells were then used in the standard assay where they detected asfew as 1000 cfu B. anthracis spores with the monoclonal antibody and10,000 cfu spores with the rabbit polyclonal, as well as 10,000 cfu F.tularensis and 1,000 cfu B. subtilis spores.

The next set of experiments demonstrated that the specificity of theassay is determined by the antibody that is used. U937 cells wereincubated with mouse anti-F. tularensis antibodies and were tested fortheir response to 105 cfu of B. anthracis spores. As shown in FIG. 38A,the cells did not respond to B. anthracis but did to 106 cfu of F.tularensis. Alternatively, cells loaded with mouse anti-B. anthracisspore antibodies did not respond to F. tularensis but did to 106 cfu ofB. anthracis spores, as shown in FIG. 38B. Furthermore, the cells didnot show any response to the 106 cfu of F. tularensis in the absence ofanti-F. tularensis antibody, as seen in FIG. 38C.

Canary: Radiological Detection

The CANARY instrument can also be used to detect radiological materials.Radiological measurements can be made by adding scintillation fluidinstead of B cells, and measuring light emitted from the scintillationfluid in response to radioactive decay. CANARY hardware has been shownto detect signal from alpha, beta, and gamma sources, and thesemeasurements compare favorably to those made using a laboratory-basedscintillation counter (FIG. 163). In this experiment, equal amounts ofvarious types of emitters were added to commercial, aqueousscintillation fluid. The tube was shaken to mix, and placed in either acommercial scintillation counter or the normal benchtop CANARYluminometer. Light output was monitored on the same laptop computerusing the same software as a normal CANARY assay. The responses ofCANARY hardware was very similar to the response of the commercialscintillation counter.

This capability (plus that of chemical and explosive detection) makesCANARY sensors very broadly useful, as one sensor can be made that candetect all chemical, biological, radiological, nuclear, and explosive(CBRNE) materials in a variety of matrices (air, liquid, surface wipes,powders, etc.). See FIG. 163. Equal amounts of various radiologicalmaterials covering all three emitter types (alpha, beta, and gamma) wereanalyzed. The response of CANARY compares favorably to a commercial,laboratory-based instrument.

Additional Methods for Detecting Chemicals and Explosives

Background: Periplasmic Binding Proteins

The chemicals used for chemical warfare agents and/or explosives (alsoreferred to herein as “CWA/E”) are too small for CANARY to detect bydirect antibody binding. However, bacteria are well equipped to detectand identify nutrients, many of which are small chemicals in the sizerange of CWA/E. CANARY can exploit a part of the bacterial nutrientdetection pathway, and be modified to detect CWA/E.

Bacteria are motile organisms, and as such actively move towardnutrients. In order to determine the location of nutrients, bacteria useperiplasmic binding proteins (PBPs) to monitor their environment. ThisPBP family has many members, each one of which binds to a specificnutrient. Using X-ray crystallography, researchers have shown that theprotein resembles a Venus' Flytrap, consisting of 2 lobes connected by ahinge. Nutrients bind in the mouth formed between the 2 lobes, and inresponse to nutrient binding in the “mouth” of the protein, the proteincloses (more accurately, its equilibrium state changes so that it ispredominantly in the closed conformation in the presence of chemicaltarget). This dramatic shape change is used to direct bacterial movementtoward nutrients.

These and other structural studies indicate that PBPs use relatively fewamino acids to actually bind to their target. Through computationaldesign, one can predict how to mutate these amino acids so that a PBPwill bind to a chemical completely different from its original target,such as the explosive TNT, the soman simulant PMPA (pinacolylmethylphosphonic acid), and the neurotransmitter serotonin (Allert etal., Proc. Natl. Acad. Sci. USA 101: 7907-7912 (2004); Looger et al.,Computational design of receptor and sensor proteins with novelfunctions. Nature 423:185-190 (2003)). Large amounts of these mutantPBPs have been produced in bacteria, and shown to bind tightly andspecifically to their new targets.

Using standard techniques, production of a high affinity CWA/E bindingprotein can be designed. If necessary, the design can start with severaldifferent parent PBPs, computationally designing all of them to bind toa given target, and testing the resulting affinities of each. Forexample, 3 different PBPs were selected as starting points to develop abinding protein for TNT: arabinose-binding protein (ABP),histidine-binding protein (HBP), and ribose-binding protein (RBP).

Published reports show that monoclonal antibodies can be readily madeagainst the closed (target-bound) form of HBP (Wolf et al., J. Biol.Chem. 269: 23051-23058 (1994); Wolf et al., J. Biol. Chem. 271:21243-21250 (1996)). These antibodies bound much faster to the HBP inthe presence of histidine, when the protein would be predominantly inthe closed conformation. In essence, then, the rate of antibody bindingto the HBP protein is a measure of the target (histidine) concentration.

All PBPs undergo a large conformational change between the open andclosed forms. Therefore, antibodies can be generated against the closedconformation of each PBP. Note also that the amino acids that aremutated to change the specificity of a given PBP are limited to thebinding pocket. Thus, it is to be expected that a single antibodyagainst the closed form of RBP, for instance, will also bind to theclosed forms of the RBP mutants that bind to TNT or PMPA. TheTNT-binding mutant could be put in “Channel 1” of the sensor, and thePMPA-binding mutant in “Channel 2”, but a single CANARY cell line thatreacts against the closed form of RBP can be used to detect targetbinding in both channels 1 and 2. The identity of the target chemicalwill be known because a different, target-specific PBP is used in eachchannel of the sensor. This means that the sensor should require farfewer CANARY cell lines than the number of chemicals that it canidentify, greatly simplifying development of reagents for additionalCWA/E.

Chemical Detection by CANARY Using Computationally Designed PBPs byCombining Individual Elements:

(1) Periplasmic binding proteins have been computationally designed thatbind to a variety of chemicals. These proteins have been produced inbacteria, isolated, and their affinities to novel targets, including TNTand PMPA, measured. (2) These PBPs undergo conformation changes in thepresence of ligand that can be measured using antibodies specific forthe closed conformation. (3) CANARY has demonstrated the capability touse antibody binding to detect protein at attomole levels. Therefore,the CANARY assay can be adapted to detect PBPs in the closedconformation (see FIG. 164). This closed conformation will indicate thepresence of CWA/E.

In detecting chemicals or explosives in the air, there are at least 2possible methods for vapor sampling. The first is impingement, in whichair is bubbled through liquid, capturing vapors and particulates. Thisis a time-tested method for air sampling. An alternate collectionstrategy is Solid phase extraction (SPE) or solid phase microextraction(SPME). This technique traps vapors directly from air onto dry,functionalized resins. Typically, these resins are eluted using heat ororganic solvents.

16 Channel Sensor Example

A new approach that reduces the time to measure multiple samples (whilekeeping the hardware requirements minimal) has been successfully tested.An experimental sensor has been designed that allows the simultaneousmeasurement of 16 samples using a single light-gathering channel. Thesensor consists of a rotor holding sixteen 1.5-ml tubes horizontally,equally distributed about its circumference, and driven by a variablespeed motor about a vertical axis (FIG. 39). A single fixedphoton-detecting element (in this case, a PMT) is positioned in theplane of the rotor just beyond the path of the tubes during rotation. Inthis way, each of the tubes is sequentially and repetitively broughtinto close proximity to the PMT, allowing its light output to be sampledon each pass. Finally, an optical switch consisting of an optical source(an infrared LED) and a detector (a phototransistor) is used to controlthe counting of detected photons and the reorganization of the data into16 fields, each associated with a specific sample.

A single measurement consists of:

1. Preparing 16 samples (and/or controls) in individual 1.5-ml tubes;

2. Introducing an aliquot of emittor cells into each of the tubes;

3. Installing the tubes into the rotor situated in a dark box;

4. Localizing the emittor cells at the bottom of the tubes using a brief(5 sec) centrifugal spin at high RCF (2000 g);

5. Reducing the rotor speed to 60 rpm for the duration of themeasurement (1-2 min), each tube being sampled once every second; and

6. Generating a time series of photon counts for each sample for displayand/or input to a computer algorithm for evaluation.

An example of the data from a 16-channel measurement, seen in FIG. 40,shows an LOD comparable to that of the single tube method. While this 16channel sensor will measure 16 samples as designed, larger samplenumbers are possible by increasing the number of channels, thoughphysical size and the statistics of sampling will ultimately dictatepractical limits. Similarly, smaller sample numbers are possible bydecreasing either the number of samples loaded onto a sensor, or byreducing the number of channels on the sensor. A CAD drawing of the16-channel portable sensor design is shown in FIG. 41.

A further implementation of this 16-channel design is referred to as aTCAN sensor. The TCAN (Triggered-CANARY) biosensor is an automatedbiosensor which combines both aerosol collection and B-cell liquiddelivery into an integrated radial disc format. The TCAN CANARY disc(CD) (FIG. 42) interfaces with a manifold assembly which splits an airflow into separate channels. The aerosol collection assembly (FIG. 43)uses dry impaction techniques to then localize particles from the airflow into the bottom of clear plastic tubes.

After impaction of aerosol particles, the CD interfaces with themanifold assembly to actuate valves located in the disc. The disc israpidly spun, which in turn causes the emittor cell liquid to deliver toindividual tubes using centrifugal force (FIG. 44). An optical detectoris then used to identify potential bioagents based on the photon outputemittor cells interacting with the aerosol particles. This process ofaerosol collection and emittor cell delivery can be repeated severaltimes in one disc. This feature allows multiple CANARY assays to beperformed after several trigger events without changing the CD.

Aerosol-Collection Techniques

Dry aerosol-collection technologies specifically tailored for the CANARYsensor have been developed to take full advantage of the potential speedof CANARY. Unlike many other air-collection systems that require wettingagents and complicated fluidics, the dry-impaction system collectsparticles directly from the air onto a dry surface thereby eliminatingalmost all consumables from the process. In addition to the low materialconsumption of this impaction system, it does not suffer from thelow-temperature freeze-out experienced by liquid-based collectionsystems.

This simple collection method separates more dense pathogen particlesfrom the airstream by exploiting the relatively high momentum ofparticles to force them to impact on a dry surface where a fraction ofthe impacted particles are bound non-specifically and retained. Thebasic concept and one of our collector prototypes are shown in FIG. 23.

An ideal aerosol impactor shows little or no collection of very smallparticles (which can follow the diverted air stream), almost 100%collection of large particles (whose momentum takes them out of the airstream), and a smooth transition in efficiency of capture for particlesizes between these extremes. Impactors are typically characterized bythe particle size at which 50% collection efficiency occurs. FIG. 165shows that for this prototype tube impactor, 50% collection efficiency(D₅₀) occurred at approximately 1-μm diameter at a flow rate of 5 litersper minute. Collection of larger total numbers of particles wasaccomplished easily by increasing the sampling rate or sampling time toincrease the total volume of air sampled.

CANARY sensors have been used to identify bioagents collected using dryimpaction without further processing since this method localizesbioagents to the tube surface, eliminating the need to pre-spin thesample for maximum performance. This allowed the CANARY assay protocolfor dry sample identification to be much faster and simpler to perform(and automate) than the protocol used for liquid samples. Identificationof dry samples also has the potential to provide improved overallsensitivity to small viruses and other pathogens that are not readilysedimentable in the liquid assay since all collected particles will beadhered to the bottom of the tube during impaction regardless of thesize of the individual pathogens incorporated in the aerosol particle.

Proof of Concept for Integrating Dry-Impaction with CANARY

To demonstrate the efficacy of the dry-impaction collection techniquefor the CANARY sensor application, individual Bacillus subtilis sporeswere aerosolized with a Collison nebulizer and collected in theprototype shown above for 30 seconds at 5 liters per minute. The B cellswere added directly to the sample-containing tube, placed in theportable CANARY apparatus, spun for 5 seconds, and the light signalquantified by PMT. The results are displayed in FIG. 166 and show thatthe direct-impaction technique yields a B-cell response that is similarin kinetics to the pre-spun liquid samples with no need for samplepreparation before analysis.

With an overall response time as short as 1 minute in thisproof-of-concept experiment (30 second collection followed by peakphoton intensity less than 30 seconds of analysis time) CANARYdemonstrated the potential to increase combined speed and sensitivityfor bioaerosol identification by more than an order of magnitudecompared to all other automated bioaerosol identification sensors. Thisdramatic performance improvement enables CANARY sensors to fill along-standing technology gap in sensor performance prevented sensitivedetection within ˜3 minutes that is needed to warn and protectpopulations from exposure to threatening bioaerosols. CANARY sensorsprovided the first (and still the only) demonstration of the potentialfor detect-to-warn (also known as detect-to-protect) biodefensecapability in a biological identification sensor. This uniquedemonstration of potential motivated the rapid development of automatedbioaerosol sensors to enable the technology to leave the laboratory andoperate in realistic environments to establish the real-world utility ofCANARY.

Automated Canary Bioaerosol Sensor Development

To demonstrate detect-to-warn capability in bioaerosol defenseapplications, the CANARY identification technology was seamlesslyintegrated with the dry aerosol collection architecture in twofirst-generation sensors, BCAN and TCAN. The BCAN sensor was designed toprovide 30 automated sampling and analysis cycles prior to reloadingwith sensitivity sufficient to detect low-concentration treats and wasextensively tested in a variety of environments to establish ROC curvescharacterizing CANARY performance and false positive rates in a varietyof realistic environments. The good performance characteristicsdemonstrated by the BCAN sensor provided the foundation that motivateddevelopment of TCAN under a separate program to demonstrate a simplifiedCANARY sensor tailored to meet the less demanding requirementsanticipated for indoor bioaerosol monitoring.

BCAN Sensor Development and Testing

The first step toward developing any automated CANARY sensor based onthe proof of concept results was to design a reliable way to combine thedry collection with a spin-enhanced CANARY assay. Furthermore, sincefluidics systems are not needed in this architecture for liquidcollection reagent delivery (as they are in all other bioaerosolidentification sensors) we focused our design efforts on cell dropletstorage and delivery without fluidics mechanisms. This unique approachof combining reagentless aerosol collection with a cell-based biosensorin an automated format enables complete elimination of a core systemthat accounts for much of the high cost, increased size and complexity,and reduced reliability of other bioaerosol identification sensorplatforms. The ultimate solution implemented for the BCAN sensorutilizes simple carriers incorporating appropriate aerosol collectionfeatures and individual aliquots of B cells stored in COTS capsules thatrelease their contents automatically during a brief spin aftercollection. The key details of this design are outlined in FIG. 167.

Each BCAN carrier contained 4 parallel mechanisms (or channels) thatprovide the four core functions necessary for CANARY analysis: Cellstorage, aerosol sampling, cell delivery, and signal transmission toPMTs. The BCAN testbed contained and automatically processed up to 25 ofthese carriers between reloading. Speed and sensitivity characteristicsfor BCAN were established using Collison nebulizer-generated Bacillussubtilis spore aerosols as a simulant for anthrax and other bioaerosolsand demonstrated that this first sensor could provide >96% probabilityof identification for bioaerosols at concentrations of ≧100 agentcontaining particles per lither of air (ACPLA) with a 3 minute totalresponse time that includes automated aerosol collection and analysis.Furthermore, this sensor was operated in a variety of indoor and outdoorlocations.

Over 13,000 tests were completed in 9 different locations spanning awide range of background conditions and the results established that thefrequency of anomalous positive signals (false positives) given by thissensor in realistic environments was similar to the frequency of falsepositives observed in the laboratory. These results togetherdemonstrated the utility of this first sensor for fast, sensitivebioaerosol identification in less than 3 minutes. Furthermore it wasdemonstrated that the collection time needed for positive identificationof a bioaerosol was proportional to the concentration of bioaerosolpresent so that total response times of less than 90 seconds werepossible for sufficiently high concentrations of bioaerosol. No otherantibody- or nucleic acid-based sensor platform has demonstrated thisspeed of response in an automated bioaerosol sensor.

An increase in the number of tests can be achieved by placing multipleB-cell lines or individual B-cell lines expressing multiple antibodiesin an individual tube, or channel. Such a system utilizing cell-line orantibody combinations minimizes hardware complexity (and size) and candetect 2^(n)−1 agents independently (where n is the number of channels)for a single-agent attack scenario. The practical limit of CANARY assaysusing multiple cell types per channel is reached with mixtures of threedifferent B-cell lines. As more than three cell types per tube are used,the signal strength at low concentrations of target falls below thedetection threshold as the probability of correct target-B cellinteractions diminishes. In addition to expanding the number of agentsthat can be identified for a given number of channels, introducing testredundancy using this approach has been used to eliminate uncorrelatedfalse positives (tests where not all of the simultaneous tests for agiven agent give positive results) and reduce the false positive ratesignificantly.

An extensive set of measurements and fieldings demonstrated BCAN'scapability to identify bioaerosols at biologically relevantconcentrations in as little as 90 seconds. This response time is anorder of magnitude faster than any other integrated bioaerosolidentification sensor and is the only demonstration of speed consistentwith the needs of detect-to-protect operation for biological defense.Perhaps even more importantly, the low false positive rates establishedfor CANARY testing in real-world situations (between 0.2% and 0.3% forsingle tests, and 0.1% or less for 2-fold or greater redundancy whilemaintaining ≧96% probability of identification) shows that thiscapability can be practically implemented into systems demanding lowfalse-alarm rates and superior speed for bioaerosol ID. While the BCANwas designed to be a powerful demonstration testbed, other sensorarchitectures offer potential advantages for customized applications.Motivated by the early successes of BCAN, TCAN sensor development wasbegun as a parallel sensor development effort to establish CANARYperformance for building protection using a customized sensor design.

TCAN Sensor Development and Testing

The TCAN is a CANARY based biosensor developed as a simple,cost-effective means for real-time monitoring of bio-aerosols in indoorbuilding environments.

This particular sensor was designed to combine both aerosol collectionand B-cell delivery into an integrated radial disc format. The disc isdesigned to interface with a manifold which separates particulate ladenairflow into four separate channels. Inertial impaction techniques arethen used to localize these particles into the bottom of cleardisposable tubes.

After collection of aerosol particles, valves located within the discare opened, and the disc is rapidly spun at 2000 RPM for 5 seconds. Thisspin step quickly drives the B-cell liquid into contact with thecollected particles using centripetal force. A single photomultipliertube (PMT) is then used to identify potential bioagents based on thephoton output of B-cells interacting with the aerosol particles as thedisc rotates. This process of aerosol collection and B-cell delivery canbe repeated several times, allowing multiple CANARY assays to beperformed in a single disc.

This CANARY sensor can deliver high confidence identification of suspectparticles in less than 3 minutes.

Panther Sensor Development and Testing

Building on the successes and lessons of the two first-generationautomated CANARY sensors, we have incorporated CANARY technology into aflexible bioaerosol sensor platform called PANTHER (Pathogen Analyzerfor Threatening Environmental Releases). The core functions of aerosolcollection and CANARY analysis were designed into a simple disk with 16channels that forms the core of the second-generation PANTHER family ofmission-specific bioaerosol identification sensors. The ultimate PANTHERsensors are intended for use individually or in networks to providesite/building protection, emergency response, rapid screening, andenvironmental monitoring. High-confidence identification of very lowconcentration bioaerosols in less than 2 minutes has been demonstratedusing the first PANTHER sensor, a portable unit referred to as the CUB,that is 37 lb., ˜1 ft³, and can ultimately be made for less than $20K.The design tested is simple and reliable: It has no fluidics, no liquidconsumables, minimal moving parts, loads like a CD player, andautomatically collects and analyzes the sample.

The CUB sensor was an outgrowth of a project initially focusing on thedevelopment of a CANARY-based sensor that could perform all of theautomated collection and analysis functions of the current bioaerosolsensor fielded by the US military—The Joint Point Biological DetectionSystem, or JBPDS. The PANTHER disk was designed to be the core of thissensor and enable 16 simultaneous tests to be performed on a singleaerosol. The development of CUB followed that initial design effort anddemonstrated the opposite end of the sensor complexity and capabilityspectrum: A small, inexpensive, portable sensor that could automaticallyprocess a single PANTHER disk. The resulting CUB sensor has beendesigned, fabricated, and tested. Preliminary results have demonstratedthat the CUB offers improved speed and sensitivity, detection of sporeaerosols at concentrations below 10 ACPLA and response times less than 2minutes including collection and identification, in a much smaller andless expensive sensor. Additional environmental testing in the sameenvironments used to characterize the BCAN bioaerosol sensor havedemonstrated that the PANTHER CUB also has a very low false positiverate in realistic environments.

Panther Cub Disk Design and Function

The disk used in PANTHER sensors performs two primary functions: 1) Itprovides specific geometries that enable it to collect aerosol particlesout of air being drawn through the disk and deposit them in a focusedlocation suitable for direct analysis using CANARY; and 2) It stores theCANARY B cells in sealed reservoirs that allow the reagent to bedispensed onto the collected aerosol particles without manualmanipulation. Two parts, a carrier body and a lid, were designed to beinjection moldable and amenable to ultrasonic welding to form thecompleted disk that is 120 mm in diameter and 6 mm tall withapproximately uniform wall thicknesses of 1 mm (FIG. 173). The preferredpolymer for the disk is polypropylene homopolymer because of it hasdemonstrated superior compatibility with B cells for long-term storage,but any other polymer with sufficient transparency (for signaltransmission from the B cells to the light sensing element) and B-cellcompatibility would also be suitable.

The carrier body has a continuous bottom with vertical walls oriented toform a plurality related feature sets in the welded disk for aerosolacceleration and collection and for liquid reagent storage and deliverythat are arrayed radially about the central axis. These features can beidentical or they can be tailored individually to enable a range ofcollector and assay functions to be provided by a single disk. An innerset of walls, FIG. 173A-feature 1, directs and accelerates the airflowtoward slits in the outer perimeter of the disk with widths and spacingsfrom the outer wall that can be tailored to provide for efficientcollection of aerosol particles at variety of flow rates usingprinciples well established in the literature (give references). Anouter set of walls, FIG. 173A-feature 2, form a continuous perimeteraround the edge of the disk and provide individual particle collectionsites with defined geometries that help collect the particlesaccelerated by the inner set of walls onto a plurality of sites withlocalized areas demonstrating increased particle density. Another set ofwalls positioned between walls A and B, FIG. 173A-feature 3, create aplurality of compartments in the welded disk that can contain liquidreagents and other materials that provide for their release anddistribution onto the collected particles during a subsequent spin orspins. The walls can be designed so that a single spin delivers storedCANARY cells from all compartments or so that multiple spins can deliverthe contents of individual compartments or subsets of compartments ondemand.

The lid has the form of is a 1 mm thick disk with two key sets offeatures. The first set of features comprises a variety of perforationsto allow introduction of liquid reagents (FIG. 173B—feature 3) andaerosol samples (FIG. 173B—feature 1 inlet, and FIG. 173B-feature 2outlets) into the assembled disk, and provides an indexing feature (JFIG. 173B—feature-4) that can be detected by an optical sensor to detectdisk orientation during readout. The second set of features comprisesraised structures on both sides that enable the lid to provide bafflesthat reduce carryover of light between adjacent channels in the disk(FIG. 173B-feature-5) (when the polymer used to form the disk contains asuitable pigment to make it opaque), provide features to enhance theultrasonic welding of the disk halves and 3) reduce the contact arearequired to form a air seal with the manifold that delivers aerosol tothe disk and removes depleted air after particle collection in the disk(circular rings just inside and outside the air outlets).

FIG. 174 illustrates how features A and B in the welded disk worktogether to direct aerosol flow and particle collection. Applying apartial vacuum using one or more suitable blowers or pumps to the arrayof triangular openings on the disk lid between the two circular ridgesdraws aerosol to be sampled into the disk through the large centralopening. The air is distributed into a plurality of channels and flowsradially outward as that channel tapers down to accelerate the aerosolas it nears the perimeter of the disk. The airflow is then forced tomake a sharp turn at the outer perimeter of the disk before being drawnout through the triangular openings. The momentum of the aerosolparticles entrained in the air sample prevents the particles fromturning with the air, and they instead impact the inner surface of thedisk at focused points that can be tailored by adjusting the geometry ofthe outer wall of the disk. A significant fraction of particles in thesize range of interest (typically between 1 and 25 μm) adhere to thesurface where they impact and are retained for subsequent CANARYanalysis. The distribution of collection in the disk was characterizedusing fluorescent 1 μm spherical polystyrene particles. These particleswere aerosolized then air was drawn through the disk at a rate of 30L/min/channel and the resulting distribution of particles was visualizedunder ultraviolet illumination. This demonstrated that the distributionof collected particles could be tailored so that the particles collectedinto two dense lines with positions determined by the location of wallsegment intersections on the collection surface. When the intersectionsare positioned to be at the maximum radius of the disk this results inreliable co-localization of the collected particles and the deliveredCANARY cells for optical signal stimulation and detection.

Adjusting the flow rate through the nozzle can enable the size range ofparticles collected in this disk design to be adjusted as needed. FIG.175 illustrates the relationship between the flow rate and the size ofparticles that are effectively transported through the disk and directedto impact onto the collection surface. At high flow rates (e.g. 30L/min) particles>1 μm in diameter have sufficient momentum to contactthe collection surface, however, the practical upper size limit for thisflow rate is 8 μm because particles≧8 μm in diameter have too muchmomentum to make the initial 90° turn that occurs as the aerosol turnsfrom the central inlet into the radial air channels that are in theplane of the disk. Reducing the airflow gradually increases the cutoffdiameter for particles that have sufficient momentum to impact thesurface but also enables larger particles to make the initial turn intothe disk and expands the overall size range of particles that can becollected at the analysis location. For example lowering the flow rateto 4 L/min adjusts the size range of collected particles to between 2.5μm and 25 μm, allowing much larger aerosol particles to be collected andanalyzed.

FIG. 176 illustrates how features C in the welded disk work together toprovide for storage and release of liquid reagents. The walls in thereagent storage zones are oriented to form a pocket that has an openingfacing the outer radius of the disk and so that appropriately positionedholes in the lid provide access and vent ports into this pocket forloading 1) a viscous plug to block the opening to the rest of the disk,followed by 2) addition of the liquid reagent (e.g. B cells) through theloading port while the air escapes through the vent hole. The additionof a short wall protruding into the reagent storage zone that separatesthe loading port from the vent port ensures complete filling of thepocket during loading. Once the loading of the viscous plug and liquidreagent is complete the access holes are covered using an adhesive tapeto seal the liquid into a pocket that remains air and liquid-tight untilrelease is desired. To release the liquid reagent the disk is spun to asufficient RPM (typically 4,000 rpm) so that the radial accelerationforces are sufficient to displace the viscous plug from its positionthereby opening the pocket toward the outside of the disk and enablingthe liquid to flow to the outer radius of the disk and cover thecollected aerosol particles. For liquids containing B cells the 5 secondspin that is used to dispense the liquid reagent is also sufficient tosediment the suspended B cells onto the outer wall of the disk andco-localize them where the aerosol particles were collected.

Following delivery of the B cells and any other liquid reagents, thespin is slowed down (typically to between 30 and 120 rpm) to enable asingle photon-sensing element (e.g. a photomultiplier tube (PMT), achannel photomultiplier (CPM) or other photon counting device) to recordsequentially the level of light emission from each channel as it passesin front of the photon sensor. The disk continues to rotate while thelight output is monitored for up to 2 minutes then the data is processedand stored by the sensor used to process the disk or by an attachedcomputer

Panther Sensor Description

The overall view of a compact sensor that has been built toautomatically process the CANARY disks is shown in FIG. 177. The sensorbody is 12″ H×12″ W×14″ D, weighs approximately 37 lbs. and has all ofthe necessary components and controls to automatically collect andanalyze aerosol samples using a single manually-loaded CANARY disk. Diskloading is accomplished by opening a drawer (FIG. 177—feature 1),placing the disk on a platform, and closing the drawer. When the sensorreceives a signal to collect and analyze a sample, the sensor begins topull air in via the inlet port (FIG. 177—feature 2), directing itthrough the disk, and then exhausting the particle-depleted air throughthe outlet port (FIG. 177—feature 3). Following aerosol collection for atime determined either by a pre-set parameter or by a signal receivedfrom an external controller, the sensor spins the disk at a speedsufficient to deliver the CANARY reagents (typically 4000 rpm) and beginthe analysis phase. During the analysis the spin is slowed down toenable the photon emission to be measured for each individual channel inthe disk as it spins in front of a single photon counting module.

The following core components (illustrated in FIG. 178) were assembledinto the first sensor and are sufficient to perform all of thecollection and analysis functions and enabled the performance describedbelow. The CANARY disk (FIG. 178—feature 4) was placed onto the motorassembly (brushless DC motor Faulhaber part#3564K012BK1155, FIG.178—feature 5) and the door was then closed to load the disk into thecustom light-tight box (FIG. 178—feature 7). Two Ametek blowers(part#150193, FIG. 178—feature 1) were connected to a custom manifold(FIG. 178—feature 6) that provided the interface with the CANARY diskand separated and directed the inlet and outlet flows as they enteredand exited the disk. When the blowers turned on the disk wasautomatically lifted into place under the manifold and the vacuum fromthe blowers held it in place and provided sufficient sealing force toensure proper aerosol flow through the disk. After the blowers turnedoff the disk dropped down automatically onto the motor assembly whichspun the disk to 4000 rpm for 5 seconds to deliver the cells then slowedthe spin to 60 rpm. That speed was maintained for 2 minutes while thechannel photomultiplier module (Perkin Elmer part# MCP 1984, FIG.178—feature 2) measured the light output from each individual channel.This entire process was controlled by an onboard computer (PC 104 fromDiamond Systems-part# ATH660-128 with a custom interface board, FIG.178—feature 3).

Panther Sensor Performance Demonstrations

To establish sensor sensitivity, test aerosols were produced byCollision nebulization of dilutions of a concentrated stock solution ofBacillus subtilis spores, sampled for 1 minute and analyzed using cellsspecific for the spores in the CUB. Approximate ACPLA levels produced byeach dilution are shown in the legend of FIG. 179. The 1:8000 dilutiongenerated a number of particles per liter that was indistinguishablefrom the chamber background produced when DI water was added to thenebulizer, but based on the general trend the concentration should be onthe order of 5 spores per liter of air. Even at this extremely lowconcentration, a 1-minute sample at 30 L/min consistently produced aneasily detectable signal with a peak intensity more than three timesgreater than the negative control.

The simulant identification data from the chamber studies was thencombined with background measurements made in typical indoor environmentover a 1-week period (>1000 tests) using cell lines specific forYersinia pestis, and Bacillus anthracis. Analysis of the resulting datademonstrated that the PANTHER CUB sensor provides better than 95%probability of detection for concentrations≧50 ACPLA with acorresponding false alarm rate of ˜0.1%. This performance provided asignificant enhancement of capability compared to the first-generationBCAN and TCAN sensor performance and can be optimized further withadditional hardware refinements and algorithm development.

Toxin Detection Example

Detection of soluble proteins can be achieved using a variety ofmethods. For example, in one method, two antibodies can be expressed inthe same emittor cell, wherein the two antibodies are each against adifferent epitope on the same molecule. The antibodies are thencrosslinked by monomeric antigen (FIG. 48). It should be pointed outthat the sorting of antibodies in the secretory pathway is idealized inthe schematic of FIG. 48. In one example, the antibodies can beheterofunctional, i.e., one antibody can have two different functionalantigen binding sites. In another example, each antibody has only onefunctional antigen binding site. This method depends on two factors: (1)multiple functional antibodies are expressed by the same emittor celland (2) two, linked epitopes are sufficient to stimulate emittor cells(although more than one of these pairs may be required to stimulate agiven cell).

In one experiment, multiple, functional antibodies were expressed in thesame emittor cell line (FIG. 49). A single cell line expressingantibodies against Bacillus anthracis and Yersinia pestis was generated.This clonal cell line reacts against both antigens with goodsensitivity. It will be understood that two antibodies against twoepitopes on the same soluble monomer can also be functionally expressed.Furthermore, two linked epitopes is sufficient to stimulate emittorcells.

A second method for detecting soluble, monomeric antigens is tocrosslink the soluble antigen to make it appear multivalent to theemittor cell (FIG. 50). This crosslinking can be done by attaching theprotein to beads, either via tags, in the case of recombinant proteins,or via antibody, as has been demonstrated for botulinum toxin Hcfragment. There are a variety of other possible methods for effectivelycrosslinking the antigen, as will be understood by those of skill in theart, including precipitation of antigen with trichloroacetic acid (TCA),heat, or ethanol, and attachment of the antigen to a solid phase vialigands, antibodies, or chemical functional groups. This crosslinkedmonomer can then be detected using emittor cells expressing antibodythat recognizes an epitope still available on the crosslinked antigen.

This second method has been demonstrated in practice, using the heavychain of botulinum toxin type A (BoNT/A Hc) as the soluble, monomerictarget protein (FIG. 51) and antibodies described in Pless et al.,Infection and Immunity (2001) 570-574. Monoclonal antibody (6E10-10)against one epitope was crosslinked to protein G-coated beads. Thesebeads were incubated with BoNT/A Hc for 3 hrs at 4° C., washed, and usedto stimulate emittor cells expressing a second antibody (6B2-2) thatrecognizes a different BoNT/A Hc epitope. The BoNT/A Hc-decorated beadseffectively stimulated the emittor cells, with an LOD of about 6 ng.Emittor cells expressing the same antibody as that used to bind theBoNT/A to the beads were not stimulated, indicating that the emittorreaction was not caused by aggregation of the target protein.

Chemical Detection Example

Chemical detection is of importance in both military and clinicalsettings. It is possible that some chemicals may have two epitopes towhich antibodies can bind independently. In such cases the methods forchemical detection would be identical to that for toxins detectionoutlined above. In many cases, however, there will not be twoindependent epitopes on the chemical of interest. In such cases it willbe necessary to modify the chemical such that it is capable ofstimulating the emittor cell. Four of these modifications are outlinedbelow.

1. Immobilize the chemical of interest on a solid support. Generateemittor cells expressing antibodies that recognize the portion of thechemical that remains available. When the density of the immobilizedchemical on the solid support is high enough, antibodies on the emittorcell surface will be immobilized close enough to each other to stimulatethe cell. This is analogous with the scheme for toxin detection shown inFIG. 50.2. First, generate peptide(s) that bind specifically to the chemical.Next, generate antibodies that bind specifically to the chemical-peptidecomplex. If the chemical-peptide complex is composed of two or moreepitopes, the complex can be detected by either of the two-antibodytechniques outlined in the section on toxin detection. If the complex isonly composed of one specific epitope, then an additional epitope, suchas digoxigenin, can be added synthetically to the peptide (FIG. 52) Thecomplex would then contain two antibody binding sites: (1) the epitopeformed by the peptide-chemical complex and (2) the digoxigenin epitope.Only in the presence of chemical would both epitopes be present. Thesetwo epitopes can then be detected by either of the two-antibodytechniques outlined in the section on toxin detection.3. Generate two peptides that specifically bind to the chemical (or toeach other in the presence of the chemical). Each of these peptides canbe synthetically tagged, such that only in the presence of chemicalwould two epitopes be bound to each other, and therefore detectable bythe emittor cell (FIG. 53). Alternatively, one or more antibodies can bemade against the peptide-chemical complex, and the presence of chemicaldetected as above using a combination of antibodies against the complex,or one antibody against the complex and one antibody against a peptidetag.4. As above, generate peptide(s) that bind specifically to the chemical,and generate antibodies that specifically bind to the peptide-chemicalcomplex. Dimerize the chemical-binding peptide, so that if the dimerbinds to two chemicals, it will contain two antibody binding sites. Thiscomplex can be detected by emittor cells expressing an antibody againstthe chemical-peptide complex.

Peptides that bind to small molecules have been isolated fromcombinatorial libraries. These molecules include porphyrin (Nakamura etal., Biosensors and Bioelectronics 2001, 16: 1095-1100) tryptophan(Sugimoto et al., 1999, 677-678) and cadmium (Mejare et al., 1998,Protein Engineering 11(6): 489-494). However, the use of proteins in theplace of peptides may yield higher affinity binders. Libraries have beenconstructed in which the binding sites have been combinatoriallydefined, and these can be used to isolate those binding to smallmolecules. Such a library using lipocalin as the starting protein hasbeen used to isolate binders to digoxigenin variants (Schlehuber andSkerra, 2002, Biophysical Chemistry 96: 213-228). This approach can beused starting with any number of other proteins, but particularly thosethat might be expected to already have some binding activity with thechemical target (for example, acetylcholinesterase, in the case of VXand Sarin).

Further Examples 1 Nucleic Acid Detection

RNA detection is advantageous to DNA detection in several respects.First, the are more copies of a given RNA per cell (prokaryotic oreukaryotic) than copies of the genome, so the signal per cell isessentially amplified. Second, the presence of RNA is often used s atest of viability. Third, detection of RNA does not require denaturationof 2 complementary strands, as in the case of dsDNA. Experiments wereperformed in a manner similar to ssDNA detection, except an RNaseinhibitor was added (RNasin Plus, Promega Corporation) (FIG. 55).Digoxigenin-labeled oligos were added to different concentrations ofRNA, incubated at 47 C for 2 minutes. CANARY cells expressing antibodyagainst digoxigenin were added, the tube spun for 5 sec, and lightoutput monitored.

Alternate Protocols

CANARY can also detect nucleic acids by directly labeling the target.For example, by performing PCR in the presence of digoxigenin-labelednucleotide, thus generating a PCR product with multiple antigensattached along its length. Likewise, rolling circle amplification can beused to incorporate label into target nucleic acid that can, in turn, bedetected by CANARY. Ligase chain reaction and its derivativesessentially dimerize oligos, and CANARY can be used to monitor thatdimerization if both oligos are labeled with one antigen each.

Toxin Detection

CANARY in its basic form is incapable of detecting monomeric antigens(FIG. 56), because the antigens are incapable of crosslinking monoclonalantibodies: as described herein, the assay must be modified. Two generalstrategies are being used to detect toxin simulants using CANARY: (1)make the toxin antigen appear polyvalent to the CANARY cell or (2) makethe antibody expressed by the CANARY cell polyclonal. For example, theprotein antigen can appear polyvalent to the CANARY cell by adsorbingthe antigen to beads, cells, or crosslinking the antigen with solubleantibody.

Initial experiments were carried out using a toxin simulant, botulinumneurotoxin Type A, heavy chain (BoNT/A Hc). The assay modification thathas thus far given the best sensitivity and speed for toxin simulantdetection by CANARY is to capture the simulant on antibody-coatedmagnetic beads, and detect the simulant-decorated bead using CANARYcells (FIG. 57). Three monoclonal antibodies that recognizenon-overlapping epitopes on the BoNT/A Hc toxin simulant; 6E10-10,6B2-2, and 6C₂₋₄ (donated by Dr Bavari and Dr. Ludwig, USAMRIID) havebeen used. Soluble 6E10-10 antibody is conjugated to protein-G labeledmagnetic beads, while the 6B2-2 antibody is expressed in CANARY cells.The 6E10-10 antibody-coated beads are incubated in solution spiked withBoNT/A Hc for 2 minutes, producing toxin simulant-decorated beads.CANARY cells are added, and the mixture spun for 5 seconds to pellet thebeads and cells. These beads present the immobilized BoNT/A Hc to theCANARY cell, crosslinking the antibodies and stimulating light emission.This technique can detect 800 pg of BoNT/A Hc (80 ng/ml) in <5 minutes(FIG. 58).

It should be noted that the sensitivity of the assay depends on thequality of the BoNT/A Hc. Lot-to-lot variability and storagecharacteristics of commercial BoNT/A Hc affect our apparent limit ofdetection (LOD). It is important in establishing the assay todemonstrate that CANARY is capable of detecting truly soluble protein.Fresh, unfrozen BoNT/A Hc gives a higher response (FIG. 59) than BoNT Hcthat has been frozen (the suggested method for storage). Centrifugationof frozen-thawed BoNT Hc further decreases the reactivity, indicatingthat aggregates form during the freeze-thaw process. BoNT/A Hc used inthese assays that has been stored frozen, is typically centrifuged uponthawing to remove aggregates. While this underrates the assaysensitivity, the interassay variation is decreased.

The bead-assay format is effective for soluble antigen screening inblood products (FIG. 60) using the whole blood preparation proceduredescribed herein. Whole blood was spiked with BoNT/A heavy chain, andthe blood briefly centrifuged through a polymer to facilitate separationof cells from soluble material. 6E10-10 antibody-coated beads were addedto the resulting supernatant, and assayed using 6B2-2 CANARY cells. Thesensitivity of the assay is similar to assays carried out in controlmedium, indicating that most interferents have been removed. Spikingplasma with an identical concentration of BoNT/A Hc after separationfrom blood cells gives a lower signal relative to samples in which theblood was spiked directly. This difference is probably an artifact ofthe blood sample preparation, not the presence of an additional CANARYinhibitor in the plasma.

BoNT/A Hc antigen spiked into urine can also be detected, although thesignal amplitude is somewhat reduced. (FIG. 61) In this experiment nopretreatment was performed. 6E10-10 coated beads were added directly tourine samples, the beads washed into CO2I, and 6B2-2 CANARY cells added.Two of three spiked urine samples (blue lines) showed significantresponses, while the third sample did not. It is not clear from thislimited dataset why the third urine sample was negative, or why thesignal amplitudes from samples in urine is lower than positive controls(gold lines).

The assay is also effective in detecting soluble antigen spiked intonasal swabs. To prepare samples for this assay, swabs are collected, thestem of the swab is trimmed and the swab end placed into a 5 micronfilter basket fitted over an eppendorf tube (FIG. 62). Control or BoNT/AHc-spiked CO2I medium is added, and the assembly capped and centrifuged.The filtered eluate, cleared of large particulates, is collected in theeppendorf and assayed using the normal bead procedure. The assay resultsfor both actual and mock swabs spiked with BoNT/Hc are very similar,indicating that no inhibitors are present in the nasal sample (FIG. 63).The lack of a CANARY response to nasal swabs without spiked antigen(CO2I) shows that there are no nonspecific stimulators present in thenasal swab sample.

Many solutions, such as orange juice or PBS/Tx-100, stimulate CANARYcells nonspecifically, so it is necessary to exchange the originalsolution containing the toxin simulant for assay medium. In addition tocrosslinking the target, the use of magnetic beads provides a simplemethod of exchanging the solution containing the simulant for cellcompatible assay medium. In the survey of food matrices, orange juicestands out as having a potential pH problem (pH=3.5) and water as havinga potential salt problem (none). Either of these characteristics couldalso affect the ability of antibody-coated beads to bind to the toxinsimulant. For these experiments, 1/7th volume (1.4 microliters) of asolution containing 560 mM NaCl, 1.4 M Hepes pH 7.9 was added to allBoNT Hc-spiked matrices and antibody-coated beads. This brings the watermatrix to a salt concentration of 80 mM final, the pH of orange juice toabout 6.5, and simultaneously introduces the antibody-conjugated beadsto initiate the binding step. At the end of a 12 minute binding step,190 μl of assay medium is added, the tube is placed on the magnet for 30seconds, and the supernatant discarded. The beads are resuspended in 50μl of assay medium, 20 μl of cells are added, the tube is spun for 5seconds to sediment the beads and CANARY cells, and light outputmonitored on a luminometer. (FIG. 64). In this graph, the valuesrepresent the peak light output normalized to background values (CANARYcells in assay medium with no antigen), so the red bar on the far rightis set to one. All other bars are maximum responses relative to thiscontrol. The response of CANARY to BoNT/A Hc diluted into orange juiceor PBS/Triton are very similar to BoNT Hc diluted into assay medium(positive control), with an LOD for in all 3 of these matrices of 80ng/ml. Milk inhibits CANARY responses by more than 5 fold. A generalizedCANARY sample preparation method can be applied to all liquid foodmatrices.

It is obviously critical to demonstrate that the assay works not onlywith toxin simulant, but also with the active BoNT/A toxin. CommercialBoNT/A was acquired and assayed using 6E10-10 beads and 6B2-2 CANARYcells (FIG. 65). The limit of detection of the assay to BoNT/A was about3.2 ng/ml or 32 pg of the toxin. It is unclear whether this improvementin assay sensitivity is due to the stability of BoNT/A compared to theBoNT/A Hc, or if there is an antigenic difference between the 2preparations. Similar types of BoNT/A detection results have been seenusing an alternate set of antibodies against BoNT/A from Dr. James Marksat UCSF. The best combination of these antibodies thus far is bead-boundS25 antibody and CANARY cells expressing Raz antibody (FIG. 66). Thereason for the lower sensitivity using this different antibody pairingis, at present, unclear.

CANARY can also detect BoNT/A spiked into whole blood (FIG. 67). Wholeblood was spiked with various concentrations of BoNT/A, and plasmaprepared as previously described. 6E10-10 antibody-coated beads wereadded to the plasma and incubated for 2 minutes. The beads were washedonce in CO2I and assayed using CANARY cells expressing the 6B2-2antibody. The limit of detection in serum drops by about 5 fold comparedto control medium, to about 16 ng/ml (160 pg).

Alternate Bead Binding Chemistries

Antibody-coated beads have also been made by biotinylating solubleantibody and attaching it to streptavidin-coated beads. Soluble antibodywas crosslinked to biotin (Pierce Biotechnology Inc) according tomanufacturer's instructions. This biotinylated antibody was bound tomagnetic streptavidin-coated beads (Dynal, Dynabeads M-280). Initialexperiments indicate that antibody conjugated to thesulfo-NHS-LC-LC-biotin gives slightly better signal than antibodyconjugated to Sulfo-NHS-LC-biotin or sulfo-NHS-biotin. (FIG. 68).6E10-10 beads produced in this way are capable of detecting solubleBoNT/A with similar sensitivity to protein G beads (FIG. 69). Multipleantibodies can be attached to the same beads using this technique,although to date the effects of binding multiple antibodies to the samebeads has been marginal (FIG. 70).

The combination of longer incubations with fewer beads does improvesensitivity (FIG. 71). Beads were diluted from their normalconcentration (about 300,000 per assay) in a 10 fold series from 1× to0.0001×. BoNT/A at 0.32 ng/ml was added, and incubated overnight. Poorsignal was seen from samples containing the normal (1×) amount of beads,but samples with 0.1× and 0.01× beads gave robust signals. Similarimprovements in sensitivity to BoNT/A are seen using protein G coatedbeads.

Additional Formats for Toxin Detection

Additional formats for CANARY detection of toxins have been envisioned,and feasibility experiments performed (see FIG. 72 for summary). Severalof these variations are thematically similar to bead capture in thatcrosslinked antigen is presented to a CANARY cell expressing onemonoclonal antibody. In approach 2, for example, the antibody-coatedbeads are replaced with CANARY cells, which are essentially live,antibody-coated beads. Two CANARY cell lines expressing antibodiesagainst different epitopes on the same toxin are incubated in solutioncontaining that toxin. One or both cells can have an emittor molecule.In some instances, both CANARY cells comprise an emittor molecule,wherein the emittor molecules are different in the different CANARYcells. In other instances, both CANARY cells comprise the same emittormolecule type. In the assay, both cells become decorated with toxin, butthe cell is not stimulated because the toxin is monomeric. The cells arecentrifuged to the bottom of the tube, where the 2 different CANARYcells present antigen to each other. This approach is effective than(LOD=50 ng or 1 μg/ml concentration), but less sensitive than toxinpresentation on beads. It may be that fixation of one of the cells priorto decoration with toxin may better restrict the movement of antibodiesin the membrane, and therefore better stimulate the opposing CANARYcell.

An alternative approach is to make a polyclonal CANARY cell (approach4). Two different antibodies are expressed in a single CANARY cell line.Because these antibodies bind to different non-overlapping epitopes onthe same toxin molecule, the CANARY cell can be stimulated directly besoluble antigen. Multiplexing studies have shown that a given CANARYcell line can express up to three different antibodies without affectingthe sensitivity of the cell to antigen, implying that expression of 2different antibodies against BoNT in the same CANARY cell line shouldnot be a problem. This would simplify the assay because a bead additionstep would not be necessary. However, sample preparation would requireexchanging the solution containing the toxin for cell assay medium.

A final approach uses the same CANARY concept, but a different cellline. In this embodiment, a single cell line is generated that expressesthe Fc receptor and aequorin. The Fc receptor binds to the Fc portion ofantibodies, leaving the antigen-binding regions free to bind to target.Soluble antibody added to these cells produces a “new” cell line withthe specificity of the added antibody in 10 minutes. Addition of antigento these cells crosslinks the Fc receptors, stimulating light emissionfrom aequorin. This approach works with both polyclonal and monoclonalantibody against Bacillus anthracis. For toxin detection, a polyclonalantibody against toxin (or 2 monoclonal antibodies against toxin) can beadded to the cell, and the Fc receptors crosslinked by soluble antigen.

Alternative Protocols:

Further improvement may be found by the addition of a third, solubleantibody to the assay. Published data from Dr. J. D. Marks' laboratory(Nowakowski et al PNAS (2002) 99(17):11346-11350) shows that incubationof BoNT/A with one monoclonal antibody increases the apparent affinityof a second monoclonal antibody against a different epitope by about 100fold. In this embodiment, a soluble antibody against a third epitope onthe BoNT/A would be added with the antibody coated beads. Binding of thethird antibody to BoNT/A would improve the kinetics of BoNT/A binding tothe beads.

Alternatively, the biotinylated antibody need not be present on thebeads when it is introduced into the assay. Soluble biotinylatedantibody and streptavidin beads could be added separately. It could bethat this will improve the binding of the antibody to the antigen, andthe high affinity of the biotin-streptavidin interaction will quicklybind the antibody-antigen complex to the beads.

The use of protein G beads or streptavidin beads is one of convenience.Any support capable of crosslinking the antibodies can be used, such asdendrimers, tube surfaces, or membranes. Antibody could be labeled withanything that will attract it to a surface from which it will be able topresent “polymerized” antigen.

Further Examples 2 Protocols for Assaying Plant Pathogens by CANARY

Plant tissue is a complex matrix which can adversely affect the CANARYassay by non-specifically inhibiting or activating the B cells.Therefore specific methods have been developed to process plant tissueto extract agents for detection by CANARY.

Bacterial Agents:

For plant bacterial pathogens which block the xylem, such as, but notlimited to, Ralstonia solanacearum, the following method is employed toextract the agent.

-   -   Crown tissue is recovered by cutting the base of the plant stem        at the soil line.    -   Compressed air, or any other method which will remove the excess        soil is used to clean off the stem    -   A second cut is made ˜1 cm from the base cut to yield a        cross-sectional piece    -   Using a circular punch slightly smaller than the diameter of the        stem, core the section to remove the outer layer (<1 mm thick)    -   Place the core into an appropriate-diameter tube containing 1 mL        of distilled water or CANARY cell assay medium (CO2I) and soak        for 5 minutes    -   Remove the core sample from the tube, vortex the liquid    -   Any portion or all of the sample can be assayed as follows:    -   Centrifuge sample at 10K-18K RCF for 2 minutes in swing-bucket        microfuge    -   If distilled water was used for the extraction, aspirate the        supernatant and discard, add 0.5 mL CO2I to the tube, vortex and        centrifuge sample atl OK-18K RCF for 2 minutes in swing-bucket        microfuge    -   If CO2I was used for the extraction, no replacement step is        required    -   Add 0.02 mL CANARY cells to assay tube, centrifuge for 5 seconds        and read signal output in luminometer.        See FIG. 100. The graph demonstrates detection of 100 cfu/mL (5        cfu/CANARY test) of Ralstonia solanacearum in geranium extract        utilizing the protocol listed above and pictured in FIG. 101.

Ralstonia spp.:

Relatively little sample prep is needed for ralstonia-infected tissue.Since the bacteria blocks the xylem (the vascular system of a plant),“bacterial streaming” (i.e. flow of the bacteria out of the cut end of astem) results when the tissue sample is placed under water. This allowsfor recovery of ralstonia from infected tissue without having to grindthe sample, thereby eliminating the need to extract the bacteria frompotentially assay-interfering plant debris.

To test a plant sample, geranium in this case, the following procedureis performed. The crown, the area of the stem just above the soil, issliced in cross-section and any residual soil is removed. A secondcross-sectional cut is made ˜1 cm above the first cut and a core samplejust slightly smaller than the diameter of the stem is taken. Thisprocess leaves the xylem intact but removes the outer covering of thestem which interferes with the CANARY assay. The core sample is thenplaced into extraction medium for 5 minutes. Because the extractionphase takes place in CANARY assay medium, additional wash steps to makethe sample compatible with CANARY are eliminated thereby shorting theprocessing time. We were able to detect ralstonia in seeded geraniumextracts, at the same level of sensitivity as ralstonia in extractionmedium alone (i.e. no plant tissue present), indicating that thepresence of plant extract does not inhibit the ralstonia-specific CANARYsignal.

A signal, clearly discernable from background (i.e. geranium extractwithout ralstonia) is apparent within 30 seconds from the time that thesample is put into the luminometer. The entire process, including sampleprep, can be completed in less than 10 minutes. The assay is capable ofdetection of as few as 5 cfu ralstonia per CANARY test. Comparableresults were obtained from the CANARY assay when eight differentisolates of live R. solanacearum R1bv1 were tested.

Viral Agents:

The potyvirus group comprises the largest and economically the mostimportant group of plant viruses. The broad spectrum-reacting monoclonalantibody, PTY1, which is expressed on CANARY B cells recognizes acryptotope (an epitope found not on the virion surface but rather oncoat protein subunits found within the intact virion). This presentsspecial issues for CANARY which requires that the cryptotopes on thevirus be exposed in order to be accessible to the B cells. The methoddescribed herein exposes the cryptotope by binding the potyvirus topristine, 1-2 micron polystyrene beads. See FIG. 156. The technologyalso works with magnetic polystyrene beads. As the virus binds to thebeads, it causes the virus coat to unwind and expose the epitope. Thebeads also provide a second advantage for the CANARY assay. Thepotyvirus is a long flexuous, filamentous particle (12×680-900 nm) whichcannot be sedimented by quick, low speed centrifugation. By attachingthe virus to beads which sediment very rapidly at a low spin speed orwhich can be concentrated with a magnet, the sensitivity of the CANARYassay for potyvirus is greatly increased. No special devices orequipment are needed to perform the sample prep/CANARY assay whichincorporates the beads.

See FIG. 102. The graph shows detection of 5 ng/mL (0.05 ng/CANARY test)of BYMV, a potyvirus, using the bead attachment process described above.The method allows for collection-to-detection in under 7 minutes. Testson six other strains of potyvirus resulted in similar limits ofdetection.

Phytophthora spp.:

Two B-cell lines to detect phytophthora, a fungal-like plant pathogen ofconsiderable economic importance, were developed. The genes for theantibodies were extracted from hybridomas, PH 3812 and PH 4831, providedby Neogen Corporation. The antibodies recognize the mycelial portion ofPhytophthora spp.

Sample prep for extraction of phytophthora is slightly more complicatedthan for the other two pathogens previously mentioned. Like tissueinfected with potyvirus, it must be ground to liberate the organism.Although phytophthora is large enough to be sedimented bycentrifugation, the plant debris co-sediments, interfering with theassay. In addition to the larger debris generated by macerating theplant tissue, abundant small particles (e.g. fines) also contaminate thesample and cannot be separated from the phytophthora by filtrationwithout concomintant loss of the pathogen. The debris interferes withthe CANARY assay by blocking light detection and in some instancescauses a non-specific signal. We again took a bead-binding approach tosample prep for extraction of phytophthora from plant tissue. Unlikepotyvirus, which has a natural affinity for polystyrene and binds veryrapidly to it without any special treatment, phytophthora will not bindto an untreated bead surface. Therefore, phytophthora mycelia werecaptured by magnetic beads coated with a second phytophthora-specificantibody (i.e. recognizes a different epitope from the antibodyexpressed on the surface of the B cell) allowing the pathogen to bepulled away from the debris. Using a magnetic “pick-pen”, the bead-boundphytophthora can be easily transferred to an assay tube and the CANARYassay can then be performed as indicated earlier. The rate-limiting stepin sample prep is the 15 minutes required to achieve sufficient bindingof the phytophthora to the antibody-coated beads.

Using this technique, we were able to demonstrate a dose dependentresponse to both live Phytophthora infestans and Phythophthora capsicimycelia, as well as detection of Phytophthora infestans in seeded potatotuber extract. LODs were not determined for the tests with phytophthora,since the antigen preps consisted of ground mycelia harvested fromactively growing phytophthora cultures. 10-fold dilutions of the groundmycelia were tested until the signal returned to baseline (nophytophthora) levels.

Protocols for Assaying Blood-Borne Pathogens by CANARY

There are many parameters that influence CANARY's ability to detectblood-borne pathogens. As with other complex matrices, blood containsboth activators and inhibitors of the CANARY assay. Light transmissionis blocked because whole blood is opaque and pathogens can be eitherintracellular or in the fluid phase of a blood sample. Additionally,variability among samples from different donors has necessitateddevelopment of a universal sample preparation method that will workregardless of donor status. Described herein are method of whole bloodsample preparation procedures and devices which overcome all of theseissues and still allow for the detection of pathogens in blood withoutsacrificing either the speed or sensitivity of the CANARY assay.

The method uses a commercially available plasma-separation tubes (PST)and differential centrifugation. This process uses a thixotropic gelwith a density between that of plasma and blood cells, which forms abarrier between the plasma and cells when the tube is centrifuged. Thebacteria or viruses present in the blood, being of lesser density thanthe gel, remain in the plasma (fluid) phase during the centrifugation.The plasma can then be harvested and tested in CANARY.

Device and Protocol for CANARY Detection of Fluid-Phase Blood-BornePathogens:

A sample device (FIG. 103) has been assembled from modified commercialoff the shelf (COTS) parts that enable the separation of whole bloodsamples in three rapid, simple steps. The device consists of acommercially available heparinized capillary PST blood collection tube.A threaded connecting collar is fitted over the cap of the PST tube,from which the top has been punched out. A stopper is then placed in thetop half of the collar. One-half milliliter of whole blood is collectedinto a heparinized plasma separation tube (step 1) and centrifuged for90 sec (step 2). The stopper is then replaced with a threaded 1.5 mLCANARY assay tube. The separated pathogen-containing plasma, withrecovered volume ranging from 50 to 250 □L, is then collected into theassay tube by inversion (step 3). The plasma is mixed with 0.5 mL ofassay medium (a process that reduces the effect of a CANARY cellactivator that is present in plasma) and the mixture is centrifuged topellet the pathogen. The sample is then tested with pathogen-specificCANARY cells as per the standard protocol. The total time required fromblood collection to pathogen detection is approximately 5 min.

Using the simple three-step procedure detailed above, the limit ofdetection of Yersinia pestis in whole blood is 1000 cfu/mL (125cfu/CANARY assay) with a total time from blood collection to agentdetection/identification in approximately 5 minutes. See FIG. 103.

Device and Protocol for CANARY Detection of Intracellular Blood-BornePathogens:

Modifications to the device developed for isolation of fluid-phasepathogens allows for the recovery of white blood cells containingintracellular pathogens, plasma and fluid-phase pathogens all in onestep. This is accomplished by incorporation of a white blood cellsisolation medium (Ficol-Diatrizoate) into the device. There is currentlyno commercial device with this configuration that is built on such asmall scale (i.e. capable of separating only 0.5 mL whole blood).Therefore the unit is assembled in its as follows.

Instead of a PST tube, an empty (no gel) capillary blood collection tubeis used as the base tube. The following components in the order thatthey are listed are then added to the tube (Note: the amounts are highlyrelevant to the device functioning properly):

-   -   200 microliters Ficol-Diatrizoate (FD    -   5 millimeters Polyester gel    -   100 microliters Phosphate Buffered Saline (PBS)    -   500 microliters Whole heparanized or EDTA blood        See FIG. 104 for the tube configuration.

The tube configuration is identical to that described for fluid-phaseseparation and the same modifications are made to accommodate thethreaded collar and assay tube described earlier. Once the blood isadded to the tube and capped, the tube is inverted several times to mixthe blood with the PBS and then centrifuged for 90 seconds. The lowerdiagram in FIG. 104 indicates the position of the componentspost-centrifugation.

The stopper is replaced with the CANARY assay tube and the cells, plasmaand any free pathogens are collected by inversion of the device.Additional steps are useful at this point compared with the fluid-phasepathogen recovery assay. The pathogen-containing white blood cellsshould be lysed to allow release of agent so that it is accessible tothe CANARY cells. First, the tube is centrifuged at 11000 RCF for 1minute to pellet the white blood cells and any free (fluid-phase)pathogens. The liquid is discarded and a commercial lysing agent isadded to the CANARY assay tube which is then vortexed to mix the cellswith the lysing agent. The tube is incubated at room temperature for 5minutes with occasional vortexing and then centrifuged again at 11000RCF for 1 minute to pellet the pathogens. The lysing reagent above thepellet is discarded and 0.5 mL of CANARY assay medium is added to thetube which is again vortexed and centrifuged. The sample is now readyfor the CANARY assay and follows the standard single sample assayformat, i.e. add the B cells, centrifuge for 5 seconds and record lightoutput in luminometer. The total time required for this assay fromcollection-to-detection is ˜12 minutes.

The limit of detection for Y. pestis in spiked whole blood is 1000cfu/mL when the blood sample is processed by the method described aboveto obtain intracellular pathogens (see FIG. 105).

Further Examples 3 CANARY B-Cell Impaction Techniques

The invention describes techniques for the efficient delivery of CANARYB-cells to wet or dry-impacted samples without centrifugation. Thesetechniques should enable simpler, cheaper automated CANARY based onminimization of moving parts and time-partitioned photon readout.

Summary Technical Description

The device incorporates techniques using droplet impaction to maximizethe rapid encounter between CANARY B-cells and the antigen-containingtargets under investigation. Several variations are described (listedbelow) and relevant experimental and analytical techniques are describedbelow.

Technique 1 “B cell Spray”

Technique 2 “CANARY Assay without Centrifuging”

Technique 3 “CANARY B cell Impaction”

Technique 4 “TCAN-3 B-cell delivery concept”

Technique 5 “Update on B-cell Impaction and CANARY”

The techniques described herein refer to either aerosolized antigen ordroplets of antigen solution impinged onto a surface through an impactorduring antigen collection. Subsequently, droplets of CANARY B-cells areaerosolized and impacted onto the same surface. The methods forimpaction are either mechanical atomization and spraying onto theimpacted antigen droplet from a fluid reservoir (Technique 1 through 3)or via the pressure differential created from a rapid puncture of aB-cell fluid reservoir (Technique 4). Technique 5 describes a series ofexperiments designed to verify the survivability of the B-cells duringsuch aerosolization schemes. In all cases, the B-cells rapidly encounterthe antigen on a transparent surface, beneath which is a photodetectoror an optical waveguide to a photodetector. Upon binding of the B-cellantibodies to the impacted antigen, light is emitted and detected by thephotodetector. The signal-to-noise ratio of the system can be improvedby matching the optical waveguide geometry to the impaction nozzlegeometry, which can be used to focus both the collected antigen as wellas the atomized B-cell solution.

This device or the methods described herein can be used to conductCANARY assays without centrifugation, thereby reducing the complexity ofan automated identification instrument and potentially improving theperformance. It uses aerosol impaction as part of a rapid immunoassay.

The CANARY assay is an extremely rapid immunoassay, with the primarytime delay resulting from the current technique of centrifuging theB-cell solution in order to provoke binding to the antigen. This methodnot only introduces a time delay but, more significantly, requiresgreater device complexity (motors, engagement and disengagementmechanisms, position and velocity encoding, etc.) than the methodproposed herein. The new technique uses aerosol impaction to bring theantibody and antigen into contact. The reduced complexity can alsoresult in smaller, less expensive automated identification sensors thancurrently exist, thus enhancing their use as part of proliferatedsensing systems.

This device can be used for the following: Biodefensedetection/identification systems, either continuous monitoring ortriggered; human health care—clinical disease and disease statecharacterization; environmental sampling and background floracharacterization; food testing; animal health, as will be understood bya person of skill in the art.

Technique 1: B Cell Spray

Goal

The goal of this experiment was to determine if spraying B cells with anatomizer would be an alternate B cell delivery mechanism. Cell volumedelivered, cell viability, and activity were measured.

Experimental Design

An alternate method of delivering a controlled volume of B cells wasinvestigated. Sprayed B cell kinetics was investigated for liquid anddry samples, and was compared to samples tested with 20 ul B cells.These experiments were tested for cell counts, viability, activityreproducibility within a concentration, and background levels. Theeffect of spinning cells after delivery, and the typical cell volumesprayed was also tested.

B cells were loaded in a 3 ml atomizer Qosina spray bottle and used todeliver cells to samples containing Ba or Yp. To determine the volume ofeach spray, the spray bottle was filled with 2 ml CO2I and one spray wasdelivered to individual eppendorf tubes until the spray bottle wasempty. The eppendorf tubes were centrifuged at 10,000 rpm for 30 secondsand volume was measured with a pipette. To measure cell counts, Ba Bcells were loaded into spray bottle and sprayed into 5 individualeppendorf tubes. The eppendorf tubes were centrifuged at 10,000 rpm for30 seconds and volume was measured with a pipette. 10 ul of cells werethen loaded into hemocytometer for counting. Cell counts were comparedto cells counted directly from original tube of cell preparation.

In order to measure B cell activity for liquid samples, 50 ul of sampleswere prepared with agent in 1.5 ml eppendorf tubes, and centrifuged at10,000 rpm for 2 min. For dried samples, 5 ul of agent, diluted inwater, was prepared in 1.5 ml eppendorf tubes, centrifuged at 10,000 rpmfor 2 min, and allowed to dry overnight. 1 spray of B cells, typicallywith a volume of 34±8 ul/spray, was directly sprayed into tube. Sampleswere then spun in a mini-centrifuge for 5 seconds and read with aBerthold luminometer.

Results

Results indicate that each spray bottle can be loaded with 2 ml of Bcells and can be sprayed 45-47 times. Each spray delivers 34±8 ul/spray(n=47). While cells counted directly from original tube average to3.2×105±8.0×10⁴ cells/ml (n=5), sprayed cells showed a reduced averageof 1.3×105±2.9×10⁴ cells/ml (n=5). Consequently, the number ofcells/sample delivered resulted in 5392±954 (n=5) for sprayed cells and5283±76 (n=5) for cells delivered with 20 ul pipette.

FIG. 106 is a graph of Ba Standard with 20 ul cell delivery. 50 ul of Basamples prepared in CO2(I) media and tested with 20 ul B cells. Resultsindicate low background and an LOD of 50 cfu Ba (n=2).

FIG. 107 is a graph of Ba B cell spray. 50 ul of Ba samples prepared inCO2 (I) media and tested with varying number of B cell sprays. Resultsindicate increased background with 2 sprays compared to 20 ul celldelivery. Number of sprays did not affect peak intensity with 50,000 cfuBa (n=1).

FIG. 108 is a graph of Ba Standard with 1-spray cell delivery. 50 ul ofBa samples prepared in CO2 (I) media and tested with one spray of Bcells. Results indicate similar backgrounds with 20 ul cell delivery andLOD of 5,000 cfu. 50 and 500 cfu Ba showed 50% chance of detection(n=2).

FIG. 109 is a graph of Ba Standard: 500 cfu Ba detection with 20 ul Bcells. 50 ul of Ba samples with 500 cfu Ba was prepared in CO2 (I) mediaand tested with 20 ul B cells. Results 100% detection of 500 cfu evenwith higher background than normally seen (n=3).

FIG. 110 is a graph of Ba B cell Spray: 500 cfu Ba detection with1-spray B cells. 50 ul of Ba samples with 500 cfu Ba was prepared in CO2(I) media and tested with 1 spray of B cells. Results indicate 50%detection of 500 cfu and a 2-3× higher background (n=14).

FIG. 111 is a graph of Ba B cell Spray: 500 cfu Ba detection with1-spray B cells and no spin. 50 ul of Ba samples with 500 cfu Ba wasprepared in CO2 (I) media and tested with 1 spray of B cells. Sampleswere not spun for 5 seconds before reading. Results indicate no cell toagent interaction resulting in 0% detection of 500 cfu Ba (n=3).

FIG. 112 is a graph of Yp B cell Spray: 500 cfu Yp detection with 20 ulB cells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2 (I)media and tested with 20 ul B cells. Results indicate a typicalbackground and 100% detection of 500 cfu Yp (n=4).

FIG. 113 is a graph of Yp B cell Spray: 500 cfu Yp detection with1-spray B cells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2(I) media and tested with 1 spray of B cells. Results indicate aslightly increased background with 100% detection of 500 cfu Yp (n=8).

FIG. 114 is a graph of Yp Standard: 500 cfu Ba detection with 20 ul Bcells. 50 ul of Yp samples with 500 cfu Yp was prepared in CO2 (I) mediaand tested with 20 ul B cells. Results 100% detection of 500 cfu with atypical background (n=7).

FIG. 115 is a graph of Yp B cell Spray: 500 cfu dried Yp detection with20 ul B cells. 5 ul of Yp samples with 500 cfu Yp was prepared in dH2O,dried overnight, and tested with 20 ul B cells. Results indicate 100%detection of 500 cfu Yp (n=10).

FIG. 116 is a graph of Yp B cell Spray: 500 cfu dried Yp detection with1-spray B cells. 5 ul of Yp samples with 500 cfu Yp was prepared indH2O, dried overnight, and tested with 1-spray B cells. Results indicatea higher background, but 100% detection of 500 cfu Yp (n=10).

Conclusion:

Results indicated that spraying B cells is a suitable method for B celldelivery. Although the cell counts decreased with spraying, the largervolume allows for similar number of cells delivered per sample. SprayingBa B cells continues to show detection capabilities with 50 and 500 cfu,but at 50% detection. It is possible that optimizing sprayingconditions, possibly with a higher concentration of B cells or newercells, this activity can be recovered. Ba B cell spraying experimentsalso indicates that the 5 second spin step is still required forappropriate B cell activity. Interestingly, Yp B cell spraying did notaffect B cell activity as much as Ba detection. Background levelsremained similar and 500 cfu Yp showed 100% detection. Effects of Bcells were also tested on liquid and dried samples. First, detection of500 cfu Yp in wet or dry formats did not change with 20 ul celldelivery. Although, backgrounds increased for sprayed cells compared to20 ul cell delivery with dried Yp samples, detection of dry 500 cfu Ypremained to show 100% detection.

These results suggest the B cells may keep similar LODs after undergoingsome pump delivery mechanisms and can withstand some of the pressuresseen in capillary or small orifice environments. Sprayed B cell deliverymay facilitate field experiments where the storing and delivery is inone piece and doesn't require pipettes.

Technique 2: Canary Assay without Centrifuging

The CANARY assay. CANARY is a fast and sensitive bio-assay. It usesmodified lines of B-cells that fluoresce upon binding with antigens.Antigen cells are either centrifuged or impacted onto a surface. ThenB-cells are centrifuged onto these cells and the fluorescence ismeasured by a luminometer. Several projects (for example BCAN and TCAN)are using CANARY for field detection of pathogens, combining aerosolcollection and impaction with the CANARY assay.

Previous CANARY Systems.

In the current versions of the CANARY field detectors, cumbersomecentrifuging equipment and delicate optical equipment are of necessitycombined in a small space. This requirement plagues the design andconstruction of these detectors. Eliminating centrifuging reduces designcosts, construction costs, and maintenance costs plus improvesreliability. We describe an alternative technique using impaction.

Alternative Techniques to Impact B-Cells.

In order to avoid the expense and design complications due tocentrifuging, several methods have been suggested as alternatives tomove the B-cells to the binding surface. These include manipulation ofmagnetic beads inside the cells, thermophoresis, electrophoresis, andacoustic manipulation. Each of these methods requires the developmentand refinement of new technologies into the CANARY system.

Proposed Technique.

Described herein is a technique that uses CANARY technology applied in anovel manner, specifically the binding of B-cells to the antigens byimpaction. This technique uses an impaction well similar to that used inBCAN or TCAN. The B-cell solution is sprayed through the antigen cellimpaction nozzle. Because of their greater mass, even though the B-cellsare in solution they still impact on the impaction surface. This isdescribed in more detail in the next section. The spray is at same flowrate as used for the bioaerosol. Therefore, the same pump used forbioaerosol collection can drive the B-cell impaction.

Physics of B-Cell Impaction.

Impaction of a particle through liquid is similar to impaction of aparticle through gas. When fluid streamlines change direction suddenlydue to physical obstructions, sufficiently massive particles in thefluid cross the streamlines and collide with the obstruction. Theunitless parameter describing the likelihood of collision is the Stokesnumber. It is the ratio of the stopping distance of a particle to thedimension of an obstacle. The Stokes number is approximately

${{Stk} \approx \frac{\tau\; U}{D}},$where U is the fluid velocity moving towards the obstacle, D is the sizeof the obstacle and τ is the particle relaxation time. The relaxationtime is a function of the particle diameter, particle density, and fluidviscosity. For fluid flowing out of a nozzle onto an impaction surface,D is the diameter of the nozzle.The equation for the particle cut-off diameter at an impaction nozzle is

${d_{50} = \left( \frac{9\;\eta\;{D^{3}\left( {Stk}_{50} \right)}}{4\rho_{p}Q} \right)^{1/2}},$where Stk₅₀ is a constant (˜0.5), η is the fluid viscosity (0.01 P forwater and 0.0002 P for air) and Q is the flow rate. For BCAN, Q is 2 lpmand D is 0.1 cm. Then the calculated d₅₀ for water is 6 microns and forair is 0.8 microns.

Therefore, the same impaction plumbing can be used both to impact aparticle in air and to impact a B-cell in solution.

The new method has several advantages. The new technique eliminates theB-cell centrifuge step. The method is fast—it takes only seconds toimpact the B-cells. There are no moving parts near the PMTs, which meansthat the PMTs will have a longer operating life and that they can bepositioned for a more sensitive signal. This detector is inexpensive andrugged compared to a centrifuge-based detector. It is easy to build bymodifying an existing BCAN or TCAN.

Technique 3: Canary B Cell Impaction

Goal:

To develop an alternate B cell delivery method for CANARY field devicesthat does not involve a centrifugation step.

Experimental Design

Earlier CANARY protocols require a 5 second centrifugation step at 500 gfor B cell delivery. However, centrifuging samples limit theeffectiveness of Canary field devices by setting severe designconstraints on an automated system which includes delicate componentssuch as B cells and PMTs. The new method described herein eliminatesextra moving parts by impacting both agent and B cells, as impacted in amanner similar to the BCAN or TCAN systems. This differs from thecurrent method in which only the agent is impacted. Consequently, theonly moving part is a valve for atomizing the B cells, which is placedat some distance from the binding surface.

In the new method, droplets of B cells are dispersed into the impactionstream. Experiments in technique 1 “B cell spray” show that B cellssurvive at least some form of atomization. Calculations in technique 2“Canary Assay without Centrifuging”, show that B cells will impactthrough an aqueous solution moving at the flow rates used in the BCAN.Because the BCAN has an impaction cut-off of 1 micron, B cell dropletswith diameters 10 micron and above will easily impact in the air flowprovided by a BCAN pump using the same flow rates. As the droplet sizeis much smaller than the BCAN nozzle, losses at the nozzle will benegligible. Whether B cells survive impaction can only be determinedexperimentally.

The new spraying method removes moving parts for the three operations ofagent impaction, B cell impaction and PMT measurement. Consequently,this simplifies the design requirements for the field device where the Bcells can be stored in a single reservoir at a distance. A simple valvemechanism at the impactor is used because the airflow does not need tobe separated from the B cell addition.

This technique requires a disperser capable of aerosolizing 10 or 20micron droplets. Collison nebulizers, the laboratory standard disperserfor bioaerosol has low efficiency for droplets greater than 5 microns.Two alternative atomizers have been considered. The first is a metereddose sprayer available from Qosina and developed for the cosmeticsindustry. ISome experiments indicate that particle sizes are 10 micronsor greater and produces a pulse of aerosol. These sprayers cost $1 each.The other type of atomizer for these particle sizes is the ultrasonicatomizer used for continuous flows. Two companies producing ultrasonicatomizer systems are Sono-tek and Sonaer. These systems cost from $7.5 kto $15 k.

An experiment to test the Qosina atomizer with a BCAN prototype is setupas follows (see FIG. 117). The disk will be placed in an impaction rig,which was built for BCAN experiments. It consists of an impactionnozzle, a well holding the impaction disk, and an outlet barb. The barbwill be connected through a tube to a rotometer, HEPA filter, and pumpto operate near 5 lpm. A tube will connect the inlet of the rig to atee, one end of which will be open to ambient air and the other to theQosina atomizer, which will contain a solution of B cells. A PMT will beplaced underneath the glass disk. Because the PMT is sensitive to light,the impaction rig and PMT will be placed in a darkbox. Dark tubing willconnect the outlet to the rotometer and the inlet to the tee. During thetest, agent simulant will be spotted onto the glass impaction disk andthe disk placed in the rig. Next the pump will be turned on and B cellssprayed from the atomizer. At this point, if the B cells surviveatomizing and impaction is sufficient quantity, a luminescence signal isexpected from the PMT. A supporting test will be to impact B cells ontoa disk free of agent, to test that the impaction alone does not causethe B cells to luminesce.

Technique 4: TCAN-3 B-Cell Delivery Concept

Goal:

Currently the TCAN-2 biosensor incorporates COTs trumpet valves tocontrol the release and delivery of B cells. This trumpet valve is bothexpensive and bulky. Additionally, a spring inside the trumpet valve hasto be removed prior to use to keep the valve open during thecentrifugation step. This technique proposes an alternative scheme for Bcell release and delivery based on a simple application of Bernoulli'sprinciple.

Concept:

The proposed concept utilizes the aerosol collection pump to aspiratethe B cells into the aerosol path from a liquid reservoir. This isaccomplished by sealing the B cell reservoir with a foil seal that isclosed during the aerosol collection. After aerosol collection, the sealis punctured, resulting in a pressure differential (ΔP) between theaerosol path and the reservoir.

This concept is based on the Bernoulli principle which states that thepressure of a fluid varies inversely with speed; therefore increases inair velocity will produce a decrease in pressure. The principles forthis concept are identical to common atomizers. Most atomizers work bygenerating an air flow over a liquid reservoir. The fast moving airdecreases the pressure at the inlet, aspirating the liquid into the airpath based on the pressure differential.

Bernoulli's Principle:

${\frac{P}{\rho} + {\frac{1}{2}V^{2}} + {gz}} = {{const}.}$P=pressure; ρ=density of fluid; V=velocityg=gravitational acceleration; z=height

Prior to puncturing the seal, the B cells should remain in the reservoirbecause the backend pressure (P₂) will equilibrate with the inletpressure (P₁) based on the ideal gas law. Assuming that the temperaturestays the same, as the fluid plug is pulled into the aerosol path thevolume of air (V₂) will also increase resulting in a decrease in thebackend pressure (P₂). The backend pressure will balance itself with theinlet pressure until the seal is broken. After the seal is broken thebackend pressure will equilibrate with the surrounding atmosphericpressure.

Ideal Gas Law:PV=nRTDesign Parameters:

There are several key experiments that need to be completed. Key designparameters include determining the ideal diameter and geometry ofreservoir channel. This diameter will affect the surface tension at theliquid-gas interface. The pressure differential due to the surfacetension in a capillary tube is as follows: (Surface tension=γ=0.073 N/mfor water)ΔP=2γ/radiusAs the radius is decreased the pressure needed to aspirate the liquidfrom the reservoir is also increased.Conclusions:

This method of B cell release and delivery will simplify the design ofthe CD currently being used in TCAN-2. This method could also decreasethe cost and size of the CD, resulting in cheaper and easier to produceparts. This technique may also be applicable to non-centrifugal B celldelivery approaches also described herein.

Technique 5: Further Experiments on B-Cell Impaction and CANARY

The method described herein targets the B cells onto the antigen byimpacting B cell droplets onto the antigen substrate. This isparticularly suitable for CANARY dry impaction. B cells are placed inthe same location as the antigen because they are placed by the samemechanism.

The excess stresses the B cells is subject to are those due toaerosolization. Specifically, stresses occur during aerosol transport,and aerosol impaction. During bioaerosol generation, cells may besubject to severe mechanical stresses and to charging. During thetransport stage, the droplet may suffer from solvent evaporation andchanges in solute concentration. These effects may lead to desiccation,oxygen toxicity and osmotic pressure imbalances. During the impactionstage, particles are once again subject to mechanical stresses. All ofthese effects may inactivate the B cell, preventing its use as anantigen detector.

B cells are not inactivated by aerosolization during FACS analysis, noris cell viability affected. During FACS/flow cytometer analysis, FACSmachines disperse cells one at a time into droplets (i.e., an aerosol)and the droplets are analyzed optically and then (optionally) collectedinto tubes for further study. B cells also survive for hours afterimpaction into dry tubes, even in the presence of ion chelators. Only10% of cells are lost after one hour. Therefore sufficient B cells forCANARY detection will impact in less than one second.

Test Study

To further study the effect of aerosolization on B cells, an antigen canbe placed at the bottom of a FACS sorter test tube. CANARY B cells canthen be processed through the FACS machine. The test tube can then beanalyzed in a luminometer for photon emission by the CANARY B cell. Anegative control would omit the antigen in the tube. In addition,impaction of antigen and CANARY cells into a tube together can betested.

Further Examples 4 16 Channel Sensor

Described herein is a refined and improved 16-channel sensor, thatprovides the same level of sensitivity as seen with a single-channelsystem (FIG. 121). This portable prototype is suitable for externalvalidation and testing. Specifically, it allows the simultaneousmeasurement of 16 samples using a single light-gathering channel. Thesensor consists of a rotor holding 16 assay tubes horizontally, equallydistributed about its circumference, and driven by a variable-speedmotor about a vertical axis. A single fixed photon-detecting element, inthis case a PMT, is positioned in the plane of the rotor just beyond thepath of the tubes during rotation. In this way, each of the tubes issequentially and repetitively brought into close proximity to the PMT,allowing its light output to be sampled on each pass. Finally, anoptical switch consisting of an optical source (an infrared LED) and adetector (a phototransistor) is used to control the counting of detectedphotons and the reorganization of the data into 16 fields, eachassociated with a specific sample.

A single measurement consists of

1. Preparing 16 samples (and/or controls) in individual assay tubes.

2. Introducing an aliquot of B cells into each of the tubes using any ofa variety of methods including, but not limited to, manual transfer,automatic transfer, capsules, or blister-packages.

3. Loading the assay tubes into the rotor.

4. Localizing the B cells at the bottom of the tubes using a brief (5sec) centrifugal spin at high relative centrifugal force (RCF) (2000 g)

5. Reducing the rotor speed to between 10 and 120 rpm for the durationof the measurement (1-2 min), each tube being sampled once perrevolution.

6. Generating a time series of photon counts for each sample for displayand/or input to a computer algorithm for evaluation.

Non-Centrifugal Assay Formats

Other assay formats that are compatible with a compact handheld sensortargeted at clinical, point-of-care, and forward-deployed applicationsare also described herein. In general, the goal during the explorationhas been to identify formats that can simplify both the CANARY assayprocedure and the hardware it requires, while maintaining as much of thespeed and sensitivity as possible. Specifically, focus has been oncharacterizing the performance of alternative assay procedures that canreduce or eliminate the requirement for centrifugation steps since theyare currently the primary driver of energy consumption and instrumentcomplexity. A number of approaches have been experimentally evaluatedtoward assay formats that employ physical manipulation of surface-boundtargets, microfluidic channels, wicking assemblies, filtration, ormagnetic bead capture. The use of lateral-flow assemblies and magneticbead capture, inter alia, are described in more detail below.

Physical Manipulation of Surface Bound Particle (a.k.a. ‘Pinhead’)Methods

This is a family of non-centrifugal methods for using CANARY B-cellsinspired by (and originally tested using) common straight pins. Inpractice, the straight pin can be replaced by any suitable solid surfacethat satisfies 3 basic criteria: 1) the surface does not stimulateB-cell calcium fluxes, 2) the surface is capable of receiving andretaining/binding target in a way that that does not alter the abilityof antibodies on the CANARY B-cells to bind the bound target, and 3) thesurface is amenable to physical manipulation to bring it into contactwith a layer of B cells (emittor cells) on the surface of a reactionvessel. Generally, particles to be tested can be collected onto the‘pinhead’ from air or liquid samples by various means (FIG. 122) andsubsequently presented to an aliquot of settled B cells (FIG. 123); ifthe collected sample includes the antigen to which those cell expressantibodies, a weak light signal would be generated and collected by asensitive luminometer.

In the centrifugal CANARY methods, particles (including bacteria, virus,or toxin) to be tested are localized at a sample site by either airimpaction (as in the BCAN) or, in the case of liquid sample, by a long(≧2 min), hard (≧10 K RCF) centrifugal ‘pre-spin’. (Either of thesesample preparations effectively concentrates the particles in a smallvolume near the sample site.) CANARY B-cells are then introduced intothe sample volume and, after a brief (≈5 sec), soft (≈500 RCF)cell-delivery spin, are driven to the sample site where they mayencounter particles. Because of the short time it takes to move the Bcells to the sample surface, these encounters happen over a short timewindow; the resulting luminous response from the B cells aresynchronized creating a more clearly identifiable signal in the form ofa recognizable pattern of detected photons.

Pinhead methods accomplish a similar concentration of particles and Bcells on or near a surface: particles to be tested are collected onto asurface (the pinhead) by various means, and that surface is physicallymaneuvered to a previously arranged thin layer of B cells(gravitationally settled, pre-spun, or grown adherent to a surface).This again results in a synchronized stimulation of the B cells,resulting in a sufficiently strong signal.

The first experimental validation of these concepts consisted of dryinga 2-μl samples containing known quantities of antigenic simulants ontopinheads and introducing these into settled (by centrifugation) aliquotsof various lines of B cells (each ‘line’ being a clonal population of Bcell expressing antibodies to a known agent or simulant). Strongresponse was observed when corresponding antigen and cell line wereused, and no signal was observed in mismatched cases (FIG. 124 shows atypical dose response).

The second experimental validation consisted of electrostatic collectionof Bs spores in a setup similar to FIG. 122( b). Using roughly similarconcentrations of Bs spores in air, a fixed air-flow speed, and varyingthe collection time, a dose response was observed when the collectionpins were introduced to tubes containing settled B cells which expressantibodies to Bs (FIG. 125).

Dual-Magnetic-Bead Assay

Described herein is an assay that takes advantage of two sets ofmagnetic beads. One set is specific for the CANARY B cells, while theother set is specific for a particular agent. These agent specific beadscould have either a general affinity for a particular agent class (e.g.gram+/−bacteria, viruses, proteins, DNA, etc.) (see for example,US2005/0118570 and U.S. patent Ser. No. 11/056,518, the teachings of allof which are incorporated herein by reference), or could have specificaffinity for a single agent. In FIG. 127, a standard CANARY assay wasrun alongside a dual-bead assay. Magnetic beads specific for Y. pestiswere mixed with a dilution series of Y. pestis agent for 5 min. After 5min the magnetic beads were pulled to the bottom of the assay tube alongwith any bound Y. pestis, and the supernatant was removed. Magneticallylabeled B cells were then added to the sample and pulled down to thebottom of the tube. Localizing agent and B cells with magnetic beads hasthus far proven to provide similar sensitivity to that ofcentrifugation.

Wicking Formats

Described herein is a CANARY assay in devices that layer wicking andfilter materials to accomplish sample fluid transport and antigenlocalization without centrifugation. The basic construction of thedevice and pictures demonstrating its ability to localize spore-sizedparticles are shown in FIGS. 128 and 129.

FIG. 130 shows the resulting CANARY signals for both standard centrifugeassays and lateral-flow assays using the same agent and cell samples.These and other experiments have shown that although B cells can be usedin a lateral-flow assay, the signal-to-noise levels tend to be lowerthan a centrifugation assay, thus lowering the overall LOD. The reducedsignal amplitude indicates that this format is less effective at eitherlocalizing antigen particles or synchronizing the presentation of the Bcells as they reach the particles on the filter material, or both.Increased background levels are also observed. These vary in intensitywith different wicking materials and flow rates, and are generallycorrelated with materials and flow rates that are expected to result inincreased mechanical stress on the B cells due to increased surfaceadhesion and liquid shear forces. Possible remedies include using Bcells selected for higher resistance to mechanical stress, using lowlevels of detergents to decrease system shear stress, decreasing thethickness and size of the capture zone to ensure all captured antigencan be seen by B cells, and decreasing sheer stress by reducing thelength of the wick strip. The initial devices used a 0.2-μm filter forcapture but can be combined with beads to capture particles smaller than0.2 μm.

Further Examples 5 Automated CANARY Bioaerosol Sensor Embodiments

Described herein is the combination of aerosol collection by inertialimpaction with CANARY identification in automated sensors to demonstratecollection and identification of airborne pathogen in as little as 90seconds. The fastest response times currently reported for otherautomated bioaerosol collection and identification devices is >18minutes, so this represents an improvement of more than one order ofmagnitude compared to the current state of the art. Two embodimentsbased on this design, the BCAN and TCAN sensors (FIG. 131) havepreviously been built and tested and described herein are the keymaterials, methods, and devices that are being incorporated into thenext-generation of the CANARY technology which we call PANTHER (PathogenAnalyzer for Threatening Environmental Releases, FIG. 131).

Key details of the core technology are described in the related figures(FIGS. 131-137) and their legends and can be summarized as follows:

1) Air containing aerosol particles to be analyzed is pulled through a4.75″ diameter disk with features that direct and accelerate the airflowthrough 16 or more channels with geometries that cause the entrainedaerosol particles to impact the surface of the disk in well-definedareas that are amenable to direct CANARY analysis.2) CANARY B cells are stored on board in 16 or more individual aliquotsthat can be automatically released using a number of availablemechanisms and delivered via a brief (less than 5 second) spin to eachof the aerosol collection sites.3) The spin forces contact between the CANARY B cells and the collectedaerosol particles and light is emitted from any samples that contain thepathogen target of the CANARY B cells. The disk is transparent to theemitted wavelength of light in the reaction zones and the emitted lightis collected and quantified using a photon-counting light detectiondevice (e.g. a photomultiplier tube).4) Multiple disks as described above are loaded into a device thatprovides for the storage, transport, processing, and analysis of thedata. Operation of this instrument will provide pathogen collection andanalysis that is capable of identification of airborne pathogens in aslittle as 90 seconds.

APPENDIX

Acronym/Symbol Definitions:

AC alternating current

AFB Air Force Base

ATP adenosine triphosphate

BAWS Biological Agent Warning Sensor

Bcl2111 Bcl2-like 11

Bmf Bcl2 modifying factor

BoNT/A botulinum neurotoxin A

BoNT/A Hc botulinum neurotoxin A heavy chain

CANARY Cellular Analysis and Notification of Antigen Risks and Yields

CCD charge-coupled device

CDC Center for Disease Control

COTS commercial off-the-shelf

CPT cell preparation tube

CRET chemical resonance energy transfer

DC direct current

DEP dielectrophoresis

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

DoD Department of Defense

EBs elementary bodies

EGFP enhanced green fluorescent protein

FcγRI Fc gamma receptor I

FMD foot-and-mouth disease

GADD45β growth-arrest and DNA-damage-inducable beta

GFP green fluorescent protein

GST glutathione transferase

HA hemagglutinin

HBSS Hanks balanced saline solution

Hells helicase, lymphoid-specific

Hist1H1c Histonel H1c

HSF1 heat-shock factor 1

IFNγ interferon gamma

LD50 lethal dose 50%

LOD limit of detection

NiCd nickel-cadmium

PBS phosphate buffered saline

PCR polymerase chain reaction

Pdcd1lg1 programmed cell death 1 ligand 1

PMT photomultiplier tube

PST plasma-separation tube

RCF relative centrifugal force

RLU relative light unit

TCA trichloroacetic acid

Tx-100-Triton X-100

USAMRIID United States Army Medical Research Institute of InfectiousDiseases

VEE Venezuelan equine encephalitis

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The relevant teachings of all the references, patents and patentapplications cited herein are incorporated herein by reference in theirentirety.

What is claimed is:
 1. A method for detecting an antigen in a samplecomprising: a) spraying emitter cells onto a sample, wherein the emittercell comprises a receptor and an emitter molecule that emits a photon inresponse to binding of a target antigen in the sample to the receptor;b) detecting photon emission, wherein photon emission is indicative ofthe antigen in the sample.
 2. The method of claim 1, wherein the emittercells are sprayed onto the sample using an atomizer.
 3. The method ofclaim 1, wherein the emitter cells are sprayed onto a liquid sample. 4.The method of claim 1, wherein the emitter cells are sprayed onto asolid surface comprising said sample.
 5. The method of claim 1, whereinthe emitter cells are sprayed onto a solid surface comprising a driedsample.
 6. The method of claim 4, wherein the solid surface is acentrifuge tube.